Hepatitis B virus mutants, reagents and methods for detection

ABSTRACT

Mutant Hepatitis B Virus (HBV) nucleic acid sequences useful for a variety of diagnostic and therapeutic applications, kits for using the HBV nucleic acid sequences, HBV immunogenic particles, and a method for producing antibodies to HBV. Also provided are methods for producing antibodies, polyclonal or monoclonal, from the HBV nucleic acid sequences.

This is a division of U.S. patent application Ser. No. 08/059,031 filedon May 7, 1993 now U.S. Pat No. 5,595,739.

BACKGROUND OF THE INVENTION

This invention relates generally to mutants of Hepatitis B Virus (HBV),and more particularly, relates to new mutants of HBV, their significancein clinical applications, their use as reagents in detection of HBVinfection and immunity and their use in vaccines.

HBV is known to cause a variety of disease states, from mild subclinicalinfection to chronic active and fulminant hepatitis. The hepatitis Bgenome is a circular, partially double stranded DNA of approximately3200 base pairs which code for seven viral proteins. P. Tiollais et al.,Nature 317:489-495 (1985). The polymerase gene completely overlaps theviral envelope genes PreS1, PreS2 and S, and partially overlaps the Xand core genes. The envelope of the hepatitis B virion consists of threeproteins and their glycosylated derivatives. These proteins, termedsmall (S), middle (M) and large (L) hepatitis B surface (HBs) proteinscontain the S gene sequence. W. H. Gerlich et al., in Viral Hepatitisand Liver Disease, F. B. Hollinger et al., eds. Williams-Wilkens,Baltimore, Md., pages 121-134 (1991). The MHBs contains the PreS2sequence (55 amino acids a.a!) and the L protein contains the PreS1sequence (108 or 119 a.a., depending on subtype) plus the PreS2sequence. Only a very small portion of the total hepatitis B surfaceantigen exists as complete virions or Dane particles. Two othermorphological forms, 22 nm spherical particles and filaments of 22 nmdiameter and variable length, lack capsid or DNA and are produced inhigh excess over HBV virions.

The core gene encodes the nucleocapsid protein (183 or 185 a.a.),hepatitis B core Antigen (HBcAg). Immediately upstream of the core geneis the precore region which consists of 87 nucleotides encoding 29 a.a.in phase with the core gene. The first 19 a.a of the precore regionserve as a signal for membrane translocation and eventual secretion ofthe precore gene product, termed HBe antigen (Ag). The function of HBeis enigmatic but may help the virus escape immune surveillance byinducing immune tolerance.

Because of the genomic compactness and the extensive functionaloverlaps, it is expected that significant constraints on DNA sequencedivergence would occur in order to maintain a genome capable ofefficient replication and transmission. The hepatitis B virus, however,shows greater mutability than previously appreciated. Similar to theHuman Immunodeficiency Virus (HIV), HBV uses reverse transcriptase (RT)as an essential step in the replication cycle. RT has poor proofreadingability, leading to a high rate of nucleotide misincorporation.Calculations suggest that this error-prone replication leads to onepoint replacement, deletion or insertion per 1000 to 100,000 nucleotidescopied. W. Carman et al., Lancet 341:349-353 (1993). Variability in thevirus was first discovered through classical subtyping studies of HBsAg.A. M. Courouce et al., Bibliotheca Haematologica 42:1 (1976).

Mutations may not be located randomly on the genome. Recent reports havedocumented the emergence of other mutations in the pre core, core andenvelope protein genes, PreS and S, which presumably give these mutantsa selective advantage over wild type (WT) in evading the immune system.

Evidence suggests that viral clearance and liver cell injury in HBVinfection are mediated by a cytotoxic T lymphocyte (CTL) response to oneor more HBV-encoded antigens expressed at the hepatocyte surface. M.Peters et al., Hepatology 13:977-994 (1991). A strong T-cell response toHBcAg and HBeAg antigens, but not to envelope antigens, was found inacute hepatitis B. Persistence of viral replication correlated with ablunted T-cell response to HBcAg. Although T cell response to viralantigen may be abrogated in chronic HBV, CTL response may persist inchronic carriers.

Also, there is evidence of ongoing humoral response in both symptomaticand asymptomatic hepatitis B carriers. High levels of anti-HBc areobserved in almost all HBV carriers. Twenty percent of random HBsAgpositive specimens have detectable HBeAg and anti-HBe, and 10-20% havedetectable but low level anti-HBs. A recent report using very sensitivedetection methods indicates that virtually all HBV patients with liverdisease and about 50% of chronic hepatitis B patients without liverdisease have demonstrable humoral immune responses specific for HBeAgand anti-HBsAg and PreS Ag. Much of the anti-HBs response, however, mayexist because of the different fine specificities of HBsAg and anti-HBsand probably is not neutralizing. These data support the theory thatthere is ongoing immune surveillance of precore, core and envelope geneproducts in chronic HBV carriers which could provide selective pressurefor the emergence of HBV variants.

The HBV envelope regions encompassing PreS1 and PreS2 and the region120-160 a.a. of S are exposed on the surface of the viral particles, andthus would be expected to be targets of immune surveillance. W. H.Gerlich et al., supra. Some S mutants described to date havesignificantly affected the antigenicity of the "a" epitope(s) which iscommon or group-specific determinant(s) of SHBs. W. Carman et al.,Gastroenterology 102:711-719 (1992). The "a" determinants are complex,conformational and dependent upon disulfide bonding among highlyconserved cysteine residues. The "a" immunoreactivity can be partiallymimmicked by cyclic synthetic peptides. Although the "a" epitope(s)traditionally had been defined by reactivity to polyclonal antisera, theuse of monoclonal antibody has shown that the "a" region consists of atleast five non-overlapping epitopes. D. Peterson et al., J. Immunol.132:920-927 (1984). Genetic variation in the "a" determinant leading toimmune escape has been described in vacinees in Italy and Japan and inliver transplant patients on monoclonal anti-a antibody therapy. See,for example, W. F. Carman et al., Lancet 336:325-329 (1990); H. Okamotoet al., Pediatric Research 32:264-268 (1992); G. McMahon et al.,Hepatology 15:757-766 (1992); H. Fujii et al., Biochem. Biophys. Res.Comm. 184:1152-1157 (1992); and T. J. Harrison et al., J. of Hepatology13:5105-5107 (1991). The most common mutant described to date is asingle nucleotide substitution leading to replacement of a glycine withan arginine (G-R 145). This mutation destroys some but not all "a"epitopes. Detection of anti-HBs with monoclonal antibody has not beenproblematic.

Other mutations in the "a" region lead to loss of subtypic or typespecific determinants, y/d and w/r. Several recent papers havedocumented the emergence of gross deletions and point mutations in thePreS1/PreS2 region suggesting that the production of these envelope geneproducts also are under immune selection in chronically infectedindividuals. HBV mutants which cannot replicate because of deletions inthe env, C or P genes have been reported in plasma from HBV carriers.All coexist with HBV forms which are replication competent. Okamoto etal. (supra) demonstrated that mutant genomes with gross deletions in thePreS/S, C and P genes derived from plasma or asymptomatic carriers maybe complemented in transient expressions system with hepatoma cells.Complementation was measured as the ability to secrete viral particleswith mutant genomes into the culture media. Interestingly, all mutantshad an intact encapsidation signal. Complementation with predecessor WTviruses, other mutants and even with HBV DNA sequences integrated intohost chromosomes was demonstrated in this in vitro system. Thus, thesuggestion that HBV mutants acting as defective interfering particlesmay attenuate WT virus replication and thereby help maintain persistenceof infection has been made.

The detection of mutants of Hepatitis B surface antigen therefore isimportant. Mutants may develop over time due to such factors as vaccineadministration or infection. The identification and detection of mutantHepatitis B Virus(es) may lead to vaccine development and detectionsystems to determine the presence of these mutants in test samples. Aneed therefore exists not only to identify these mutant strains, butalso to provide detection systems capable of determining the presence ofthe mutant in a test sample. A further need also exists for thedevelopment of a vaccine to such mutant strain(s).

SUMMARY OF THE INVENTION

This invention describes a mutant Hepatitis B Virus having a modified"a" determinant in which there is an insertion of two amino acids at the122 position of the HBsAg sequence, which insertion corresponds to a sixnucleotide insertion at the 366 position of the HBsAg genome. The twoamino acids inserted at position 122 are N and T, while thecorresponding nucleotide sequence appearing at position 366 of thenucleotide sequence is -A-A-C-A-C-A-.

This invention provides a purified polynucleotide of mutant HBVcomprising a modified "a" determinant in which there is an insertion ofat least six nucleotides at positions 369 of the HBsAg genome, arecombinant polynucleotide of mutant HBV comprising a modified "a"determinant in which there is an insertion of at least two amino acidsat position 122 of the HBsAg sequence, host cells and recombinantvectors reciting this insertion. The two amino acids inserted atposition 122 are N and T, while the corresponding nucleotide sequenceappearing at position 366 of the nucleotide sequence is -A-A-C-A-C-A-.The recombinant polynucleotide of mutant HBV comprises a sequencederived from a mutant HBV genome. Also, the recombinant polynucleotideof mutant HBV comprises an epitope of mutant HBV. The invention alsoprovides a recombinant expression system comprising an open readingframe of DNA derived from the genome of mutant HBV, wherein the openreading frame is operably linked to a control sequence compatible with adesired host, cells transformed with said recombinant expression systemand polypeptides produced by said cells. The invention further providespurified mutant HBV, which can comprise a preparation of mutant HBVpolypeptide. All embodiments recite the modification of the "a"determinant having either at least a two-amino insertion at aminoa acid122 or a six nucleotide insertion at 366.

Additionally, the present invention provides a recombinant polypeptidecomprising a sequence derived from a mutant HBV genome, and arecombinant polypeptide comprising a mutant HBV epitope. Also providedis an antibody directed against at least one epitope of mutant HBV.Theantibody is polyclonal or monoclonal. The invention further provides afusion polypeptide comprising a polypeptide of mutant HBV.

The invention also provides a particle that is immunogenic againstmutant HBV infection, comprising a non-mutant HBV polypeptide having anamino acid sequence capable of forming a particle when said sequence isproduced in a eukaryotic or prokaryotic host, and an epitope of mutantHBV, a polynucleotide probe for mutant HBV, and various test kits forperforming various methods to detect either mutant HBV antigen or mutantHBV antibody.

Moreover, the invention provides a method for producing a polypeptidecontaining an epitope of mutant HBV comprising incubating host cellstransformed with an expression vector containing a sequence encoding apolypeptide containing an epitope of HBV, under conditions and for atime which allows expression of said polypeptide.

Further, a method for detecting mutant HBV nucleic acids in a testsample suspected of containing mutant HBV is provided, wherein themethod comprises reacting the test sample with a probe for apolynucleotide of mutant HBV under conditions and for a time whichallows the formation of a complex between the probe and the nucleic acidof mutant HBV in the test sample; and detecting the complex whichcontains the probe. An additional method for detecting mutant HBVantigen in a test sample suspected of containing mutant HBV comprisescontacting a test sample with an antibody directed against mutant HBVantigen to be detected for a time and under conditions sufficient toallow the formation of antibody/antigen complexes; and detecting saidcomplex containing the antibody. Still another method for detectingmutant HBV antibodies in a test sample suspected of containing saidantibodies, comprises contacting the test sample with a probepolypeptide wherein said polypeptide contains a mutant HBV epitope, fora time and under conditions sufficient to allow antigen/antibodycomplexes to form; and detecting said complexes which contain the probepolypeptide.

A vaccine for treatment of mutant HBV infection also is provided whichcomprises a pharmacologically effective dose of an immunogenic mutantHBV polypeptide which contains an epitope of mutant HBV in apharmaceutically acceptable excipient. The vaccine for treatment ofmutant HBV infection also can comprise an inactivated or attenuatedmutant HBV in a pharmacologically effective dose in an pharmaceuticallyacceptable excipient.

Further, the invention provides tissue culture grown cells infected withmutant HBV and a method for producing antibodies to mutant HBVcomprising administering to an individual an isolated immunogenicpolypeptide containing an epitope of mutant HBV in an amount sufficientto produce an immune response.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides characterization of a newly ascertainedmutant of HBV that has a two amino acid (N-T) insertion at position 122of the HBV envelope region. The present invention provides methods fordetermining the presence of the mutant HBV in a test sample, andreagents useful in these methods. All aspects provide a modification ofthe "a" determinant in which there is an insertion of two amino acids atposition 122 of the HBsAg sequence, which corresponds to a sixnucleotide insertion at position 366 of the HBsAg genome.

The nucleic acid sequence derived from mutant HBV, or a portion thereofare useful as probes to determine the presence of mutant HBV in testsamples. The sequence also makes available polypeptide sequences ofmutant HBV antigen(s) encoded within the genome(s) of such mutant HBVand permits the production of polypeptides which are useful as standardsor reagents in diagnostic tests and/or as components of vaccines.Monoclonal and polyclonal antibodies directed against an epitopecontained within these polypeptide sequences, also are useful fordiagnostic tests as well as therapeutic agents, for screening ofantiviral agents, and for the isolation of the mutant HBV from whichthese nucleic acid sequences are derived.

According to one aspect of the invention, there will be provided apurified polynucleotide of mutant HBV, a recombinant polynucleotide ofmutant HBV, a recombinant polynucleotide comprising a sequence derivedfrom a genome of mutant HBV; a recombinant polypeptide encoding anepitope of mutant HBV; a recombinant vector containing any of the abovedescribed recombinant polypeptides, and a host cell transformed with anyof these vectors. All aspects provide a modification of the "a"determinant in which there is an insertion of two amino acids atposition 122 of the HBsAg sequence, which corresponds to a sixnucleotide insertion at position 366 of the HBsAg genome.

In another aspect of the invention there will be provided purifiedantigen of mutant HBV; a preparation of polypeptides from the purifiedmutant HBV; a purified polypeptide of mutant HBV; a purified polypeptidecomprising an epitope which is immunologically identifiable with anepitope contained in mutant HBV.

In yet another aspect of the invention there will be provided arecombinant expression system comprising an open reading frame (ORF) ofDNA derived from a mutant HBV genome, wherein the ORF is operably linkedto a control sequence compatible with a desired host, a cell transformedwith the recombinant expression system, and a polypeptide produced bythe transformed cell.

Additional aspects of the present invention include a recombinantpolypeptide of mutant HBV, a recombinant polypeptide comprised of asequence derived from a genome of mutant HBV; a recombinant polypeptidecomprised of an epitope of mutant HBV and a fusion polypeptide comprisedof a polypeptide of mutant HBV.

The present invention also provides methods for producing a monoclonalantibody which specifically binds to at least one epitope of mutant HBV;a purified preparation of polyclonal antibodies which specifically bindto at least one mutant HBV epitope; and methods for using theseantibodies, which include diagnostic, prognostic and therapeutic uses.

In still another aspect of the invention there will be provided aparticle which is immunogenic against mutant HBV infection comprising anon-mutant HBV polypeptide having an amino acid sequence capable offorming a particle when said sequence is produced in a eukaryotic host,and an epitope of mutant HBV.

A polynucleotide probe for mutant HBV also will be provided.

The present invention provides kits containing reagents which can beused for the detection of the presence and/or amount of polynucleotidesderived from mutant HBV, such reagents comprising a polynucleotide probecontaining a nucleotide sequence from mutant HBV of about 8 or morenucleotides in a suitable container and which nucleotides encode for theinsertion of at least two amino acids at position 122; a reagent fordetecting the presence and/or amount of a mutant HBV antigen comprisingan antibody directed against the mutant HBV antigen to be detected in asuitable container; a reagent for detecting the presence and/or amountof antibodies directed against a mutant HBV antigen comprising apolypeptide containing an epitope of mutant HBV present in the mutantHBV antigen, provided in a suitable container. Other kits for variousassay formats also are provided by the present invention as describedherein.

Other aspects of the present invention include a polypeptide comprisingan epitope of mutant HBV attached to a solid phase and an antibody to anepitope of mutant HBV attached to a solid phase. Also included aremethods for producing a polypeptide containing an epitope to mutant HBVcomprising incubating host cells transformed with an expression vectorcontaining a sequence encoding a polypeptide containing an epitope ofmutant HBV under conditions which allow expression of the polypeptide,and a polypeptide containing an epitope of mutant HBV produced by thismethod.

The present invention also provides assays which utilize the recombinantor synthetic polypeptides provided by the invention, as well as theantibodies described herein in various formats, any of which may employa signal generating compound in the assay. Assays which do not utilizesignal generating compounds to provide a means of detection also areprovided. All of the assays described generally detect either antigen orantibody, or both, and include contacting a test sample with at leastone reagent provided herein to form at least one antigen/antibodycomplex and detecting the presence of the complex. These assays aredescribed in detail herein.

Vaccines for treatment of mutant HBV infection comprising an immunogenicpeptide containing a mutant HBV epitope, or an inactivated preparationof mutant HBV, or an attenuated preparation of mutant HBV are includedin the present invention. Also included in the present invention is amethod for producing antibodies to mutant HBV comprising administeringto an individual an isolated immunogenic polypeptide containing anepitope of mutant HBV in an amount sufficient to produce an immuneresponse in the inoculated individual.

Also provided by the present invention is a tissue culture grown cellinfected with mutant HBV.

Definitions

Note: All definitions include the modification of the "a" determinant inwhich there is an insertion of two amino acids at position 122 of theHBsAg sequence, which corresponds to a six nucleotide insertion atposition 366 of the HBsAg genome. The term "mutant HBV" means a viralisolate having this modification of the "a" determinant.

A polynucleotide "derived from" a designated sequence,from the mutantHBV genome refers to a polynucleotide sequence which is comprised of asequence of approximately at least about 6 nucleotides, is preferably atleast about 8 nucleotides, is more preferably at least about 10-12nucleotides, and even more preferably is at least about 15-20nucleotides corresponding, i.e., homologous to or complementary to, aregion of the designated nucleotide sequence. Preferably, the sequenceof the region from which the polynucleotide is derived is homologous toor complementary to a sequence which is unique to the mutant HBV genome.Whether or not a sequence is complementary to or homologous to asequence which is unique to a mutant HBV genome can be determined bytechniques known to those skilled in the art. Comparisons to sequencesin databanks, for example, can be used as a method to determine theuniqueness of a designated sequence.

The derived polynucleotide will not necessarily be derived physicallyfrom the nucleotide sequence of mutant HBV, but may be generated in anymanner, including but not limited to chemical synthesis, replication orreverse transcription or transcription, which are based on theinformation provided by the sequence of bases in the region(s) fromwhich the polynucleotide is derived. In addition, combinations ofregions corresponding to that of the designated sequence may be modifiedin ways known in the art to be consistent with an intended use.

A polypeptide or amino acid sequence derived from a designated nucleicacid sequence or from the mutant HBV genome refers to a polypeptidehaving an amino acid sequence identical to that of a polypeptide encodedin the sequence, or a portion thereof wherein the portion consists of atleast 2 to 5 amino acids, and more preferably at least 8 to 10 aminoacids, and even more preferably 15 to 20 amino acids, or which isimmunologically identifiable with a polypeptide encoded in the sequence.

A "recombinant protein" ("recombinant polynucleotide") as used hereinmeans at least a polypeptide of genomic, semisynthetic or syntheticorigin which by virtue of its origin or manipulation is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature or in the form of a library and/or is linked to apolynucleotide other than that to which it is linked in nature. Arecombinant or derived polypeptide is not necessarily translated from adesignated nucleic acid sequence of mutant HBV or from a mutant HBVgenome. It also may be generated in any manner, including chemicalsynthesis or expression of a recombinant expression system, or isolationfrom mutated HBV.

The term "polynucleotide" as used herein means a polymeric form ofnucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, the term includes double- and single-stranded DNA,as well as double- and single-stranded RNA. It also includesmodifications, either by methylation and/or by capping, and unmodifiedforms of the polynucleotide.

"Purified viral polynucleotide" refers to a genome of mutant HBV orfragment thereof which is essentially free, i.e., contains less thanabout 50%, preferably less than about 70%, and even more preferably,less than about 90% of polypeptides with which the viral polynucleotideis naturally associated. Techniques for purifying viral polynucleotidesare well known in the art and include, for example, disruption of theparticle with a chaotropic agent, and separation of thepolynucleotide(s) and polypeptides by ion-exchange chromatography,affinity chromatography, and sedimentation according to density. Thus,"purified viral polypeptide" means a polypeptide of mutant HBV orfragment thereof which is essentially free, that is, contains less thanabout 50%, preferably less than about 70%, and even more preferably,less than about 90% of of cellular components with which the viralpolypeptide is naturally associated. Methods for purifying are known tothe routineer.

"Polypeptide" as used herein indicates a molecular chain of amino acidsand does not refer to a specific length of the product Thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term however is not intended to refer topost-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like.

"Recombinant host cells," "host cells," "cells," "cell lines," "cellcultures," and other such terms denoting microorganisms or highereucaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transfer DNA, and include the original progeny of the originalcell which has been transfected.

As used herein "replicon" means any genetic element, such as a plasmid,a chromosome, a virus, that behaves as an autonomous unit ofpolynucleotide replication within a cell. That is, it is capable ofreplication under its own control.

A "vector" is a replicon in which another polynucleotide segment isattached, such as to bring about the replication and/or expression ofthe attached segment.

The term "control sequence" refers to polynucleotide sequences which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, such control sequences generally includepromoter, ribosomal binding site and terminators; in eukaryotes, suchcontrol sequences generally include promoters, terminators and, in someinstances, enhancers. The term "control sequence" thus is intended toinclude at a minimum all components whose presence is necessary forexpression, and also may include additional components whose presence isadvantageous, for example, leader sequences.

"Operably linked" refers to a situation wherein the components describedare in a relationship permitting them to function in their intendedmanner. Thus, for example, a control sequence "operably linked" to acoding sequence is ligated in such a manner that expression of thecoding sequence is achieved under conditions compatible with the controlsequences.

The term "open reading frame" or "ORF" refers to a region of apolynucleotide sequence which encodes a polypeptide; this region mayrepresent a portion of a coding sequence or a total coding sequence.

A "coding sequence" is a polynucleotide sequence which is transcribedinto mRNA and/or translated into a polypeptide when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a translation start codon at the5'-terminus and a translation stop codon at the 3'-terminus. A codingsequence can include, but is not limited to, mRNA and recombinantpolypeptide sequences.

The term "immunologically identifiable with/as" refers to the presenceof epitope(s) and polypeptide(s) which also are present in and areunique to the designated polypeptide(s), usually mutant HBV proteins.Immunological identity may be determined by antibody binding and/orcompetition in binding. These techniques are known to the routineer andalso are described herein. The uniqueness of an epitope also can bedetermined by computer searches of known data banks, such as Genbank,the polynucleotide sequences which encode the epitope, and by amino acidsequence comparisons with other known proteins.

As used herein, "epitope" means an antigenic determinant of apolypeptide. Conceivably, an epitope can comprise three amino acids in aspatial conformation which is unique to the epitope. Generally, anepitope consists of at least five such amino acids, and more usually, itconsists of at least 8 to 10 amino acids. Methods of examining spatialconformation are known in the art and include, for example, x-raycrystallography and two-dimensional nuclear magnetic resonance.

A polypeptide is "immunologically reactive" with an antibody when itbinds to an antibody due to antibody recognition of a specific epitopecontained within the polypeptide. Immunological reactivity may bedetermined by antibody binding, more particularly by the kinetics ofantibody binding, and/or by competition in binding using ascompetitor(s) a known polypeptide(s) containing an epitope against whichthe antibody is directed. The methods for determining whether apolypeptide is immunologically reactive with an antibody are known inthe art.

As used herein, the term "immunogenic polypeptide containing a mutantHBV epitope" means naturally occurring polypeptides of mutant HBV orfragments thereof, as well as polypeptides prepared by other means, forexample, chemical synthesis or the expression of the polypeptide in arecombinant organism.

The term "transformation" refers to the insertion of an exogenouspolynucleotide into a host cell, irrespective of the method used for theinsertion. For example, direct uptake, transduction, or f-mating areincluded. The exogenous polynucleotide may be maintained as anon-integrated vector, for example, a plasmid, or alternatively, may beintegrated into the host genome.

"Treatment" refers to prophylaxis and/or therapy.

The term "individual" as used herein refers to vertebrates, particularlymembers of the mammalian species and includes but is not limited todomestic animals, sports animals, primates and humans; more particularlythe term refers to chimpanzees and humans.

The term "plus strand" as used herein denotes a nucleic acid thatcontains the sequence that encodes the polypeptide. The term "minusstrand" denotes a nucleic acid, that contains a sequence that iscomplementary to that of the "plus" strand.

"Positive stranded genome" of a virus denotes that the genome issingle-stranded and encodes a viral polypeptide(s).

The term "antibody containing body component" (or test sample) refers toa component of an individual's body which is the source of theantibodies of interest These components are well known in the art. Thesesamples include biological samples which can be tested by the methods ofthe present invention described herein and include human and animal bodyfluids such as whole blood, serum, plasma, cerebrospinal fluid, urine,lymph fluids, and various external secretions of the respiratory,intestinal and genitorurinary tracts, tears, saliva, milk, white bloodcells, myelomas and the like, biological fluids such as cell culturesupernatants, fixed tissue specimens and fixed cell specimens.

"Purified mutant HBV" refers to a preparation of mutant HBV which hasbeen isolated from the cellular constituents with which the virus isnormally associated, and from other types of viruses which may bepresent in the infected tissue. The techniques for isolating viruses areknown to those skilled in the art and include, for example,centrifugation and affinity chromatography. A method for the preparationof purified HBV is described herein.

General Uses

After preparing recombinant proteins, synthetic peptides, or purifiedviral polypeptides of choice as described by the present invention, therecombinant or synthetic peptides can be used to develop unique assaysas described herein to detect either the presence of antigen or antibodyto mutant HBV. These compositions also can be used to develop monoclonaland/or polyclonal antibodies with a specific recombinant protein orsynthetic peptide which specifically bind to the immunological epitopeof mutant HBV which is desired by the routineer. Also, it iscontemplated that at least one polynucleotide of the invention can beused to develop vaccines by following methods known in the art.

It is contemplated that the reagent employed for the assay can beprovided in the form of a kit with one or more containers such as vialsor bottles, with each container containing a separate reagent such as amonoclonal antibody, or a cocktail of monoclonal antibodies, or apolypeptide (either recombinant or synthetic) employed in the assay.

"Solid phases" ("solid supports") are known to those in the art andinclude the walls of wells of a reaction tray, test tubes, polystyrenebeads, magnetic beads, nitrocellulose strips, membranes, microparticlessuch as latex particles, and others. The "solid phase" is not criticaland can be selected by one skilled in the art. Thus, latex particles,microparticles, magnetic or non-magnetic beads, membranes, plastictubes, walls of microtiter wells, glass or silicon chips and sheep redblood cells are all suitable examples. Suitable methods for immobilizingpeptides on solid phases include ionic, hydrophobic, covalentinteractions and the like. A "solid phase", as used herein, refers toany material which is insoluble, or can be made insoluble by asubsequent reaction. The solid phase can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor which has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid phase and which has the ability to immobilize the capturereagent through a specific binding reaction. The receptor moleculeenables the indirect binding of the capture reagent to a solid phasematerial before the performance of the assay or during the performanceof the assay. The solid phase thus can be a plastic, derivatizedplastic, magnetic or non-magnetic metal, glass or silicon surface of atest tube, microtiter well, sheet, bead, microparticle, chip, and otherconfigurations known to those of ordinary skill in the art.

It is contemplated and within the scope of the invention that the solidphase also can comprise any suitable porous material with sufficientporosity to allow access by detection antibodies and a suitable surfaceaffinity to bind antigens. Microporous structures are generallypreferred, but materials with gel structure in the hydrated state may beused as well. Such useful solid supports include:

natural polymeric carbohydrates and their synthetically modified,cross-linked or substituted derivatives, such as agar, agarose,cross-linked alginic acid, substituted and cross-linked guar gums,cellulose esters, especially with nitric acid and carboxylic acids,mixed cellulose esters, and cellulose ethers; natural polymerscontaining nitrogen, such as proteins and derivatives, includingcross-linked or modified gelatins; natural hydrocarbon polymers, such aslatex and rubber; synthetic polymers which may be prepared with suitablyporous structures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkaineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer. All of these materials maybe used in suitable shapes, such as films, sheets, or plates, or theymay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

The porous structure of nitrocellulose has excellent absorption andadsorption qualities for a wide variety of reagents including monoclonalantibodies. Nylon also possesses similar characteristics and also issuitable. It is contemplated that such porous solid supports describedhereinabove are preferably in the form of sheets of thickness from about0.01 to 0.5 mm, preferably about 0.1 mm. The pore size may vary withinwide limits, and is preferably from about 0.025 to 15 microns,especially from about 0.15 to 15 microns. The surfaces of such supportsmay be activated by chemical processes which cause covalent linkage ofthe antigen or antibody to the support. The irreversible binding of theantigen or antibody is obtained, however, in general, by adsorption onthe porous material by poorly understood hydrophobic forces. Suitablesolid supports also are described in U.S. patent application Ser. No.227,272, which is incorporated herein by reference.

The "indicator reagent" comprises a "signal generating compound" (label)which is capable of generating a measurable signal detectable byexternal means conjugated (attached) to a specific binding member formutant HBV. "Specific binding member" as used herein means a member of aspecific binding pair. That is, two different molecules where one of themolecules through chemical or physical means specifically binds to thesecond molecule. In addition to being an antibody member of a specificbinding pair for mutant HBV, the indicator reagent also can be a memberof any specific binding pair, including either hapten-anti-haptensystems such as biotin or anti-biotin, avidin or biotin, a carbohydrateor a lectin, a complementary nucleotide sequence, an effector or areceptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitoror an enzyme, and the like. An immunoreactive specific binding membercan be an antibody, an antigen, or an antibody/antigen complex that iscapable of binding either to mutant HBV as in a sandwich assay, to thecapture reagent as in a competitive assay, or to the ancillary specificbinding member as in an indirect assay.

The various "signal generating compounds" (labels) contemplated includechromogens, catalysts such as enzymes, luminescent compounds such asfluorescein and rhodamine, chemiluminescent compounds, radioactiveelements, and direct visual labels. Examples of enzymes include alkalinephosphatase, horseradish peroxidase, beta-galactosidase, and the like.The selection of a particular label is not critical, but it will becapable of producing a signal either by itself or in conjunction withone or more additional substances.

Other embodiments which utilize various other solid phases also arecontemplated and are within the scope of this invention. For example,ion capture procedures for immobilizing an immobilizable reactioncomplex with a negatively charged polymer, described in co-pending U.S.patent application Ser. No. 150,278 corresponding to EP publication0326100 and U.S. patent application Ser. No. 375,029 (EP publication no.0406473), each of which are incorporated herein by reference, can beemployed according to the present invention to effect a fastsolution-phase immunochemical reaction. An immobilizable immune complexis separated from the rest of the reaction mixture by ionic interactionsbetween the negatively charged poly-anion/immune complex and thepreviously treated, positively charged porous matrix and detected byusing various signal generating systems previously described, includingthose described in chemiluminescent signal measurements as described inco-pending U.S. patent application Ser. No. 921,979 corresponding to EPOPublication No. 0 273,115, which is incorporated herein by reference.

Also, the methods of the present invention can be adapted for use insystems which utilize microparticle technology including in automatedand semi-automated systems wherein the solid phase comprises amicroparticle. Such systems include those described in pending U.S.patent application Ser. Nos. 425,651 and 425,643, which correspond topublished EPO applications Nos. EP 0 425 633 and EP 0 424 634,respectively, which are incorporated herein by reference.

The use of scanning probe microscopy (SPM) for immunoassays also is atechnology to which the monoclonal antibodies of the present inventionare easily adaptable. In scanning probe microscopy, in particular inatomic force microscopy, the capture phase, for example, at least one ofthe monoclonal antibodies of the invention, is adhered to a solid phaseand a scanning probe microscope is utilized to detect antigen/antibodycomplexes which may be present on the surface of the solid phase. Theuse of scanning tunnelling microscopy eliminates the need for labelswhich normally must be utilized in many immunoassay systems to detectantigen/antibody complexes. Such a system is described in pending U.S.patent application Ser. No. 662,147, which is incorporated herein byreference.

The use of SPM to monitor specific binding reactions can occur in manyways. In one embodiment, one member of a specific binding partner(analyte specific substance which is the monoclonal antibody of theinvention) is attached to a surface suitable for scanning. Theattachment of the analyte specific substance may be by adsorption to atest piece which comprises a solid phase of a plastic or metal surface,following methods known to those of ordinary skill in the art. Or,covalent attachment of a specific binding partner (analyte specificsubstance) to a test piece which test piece comprises a solid phase ofderivatized plastic, metal, silicon, or glass may be utilized. Covalentattachment methods are known to those skilled in the art and include avariety of means to irreversibly link specific binding partners to thetest piece. If the test piece is silicon or glass, the surface must beactivated prior to attaching the specific binding partner. Activatedsilane compounds such as triethoxy amino propyl silane (available fromSigma Chemical Co., St. Louis, Mo.), triethoxy vinyl silane (AldrichChemical Co., Milwaukee, Wis.), and (3-mercapto-propyl)-trimethoxysilane (Sigma Chemical Co., St. Louis, Mo.) can be used to introducereactive groups such as amino-, vinyl, and thiol, respectively. Suchactivated surfaces can be used to link the binding partner directly (inthe cases of amino or thiol) or the activated surface can be furtherreacted with linkers such as glutaraldehyde, bis (succinimidyl)suberate, SPPD 9 succinimidyl 3- 2-pyridyldithio! propionate), SMCC(succinimidyl-4- N-maleimidomethyl! cyclohexane-1-carboxylate), SIAB(succinimidyl 4-iodoacetyl! aminobenzoate), and SMPB (succinimidyl 4-1-maleimidophenyl! butyrate) to separate the binding partner from thesurface. The vinyl group can be oxidized to provide a means for covalentattachment. It also can be used as an anchor for the polymerization ofvarious polymers such as poly acrylic acid, which can provide multipleattachment points for specific binding partners. The amino surface canbe reacted with oxidized dextrans of various molecular weights toprovide hydrophilic linkers of different size and capacity. Examples ofoxidizable dextrans include Dextran T-40 (molecular weight 40,000daltons), Dextran T-1 10 (molecular weight 110,000 daltons), DextranT-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight2,000,000 daltons (all of which are available from Pharmacia), or Ficoll(molecular weight 70,000 daltons (available from Sigma Chemical Co., St.Louis, Mo.). Also, polyelectrolyte interactions may be used toimmobilize a specific binding partner on a surface of a test piece byusing techniques and chemistries described by pending U.S. patentapplications Ser. No. 150,278, filed Jan. 29, 1988, and Ser. No.375,029, filed Jul. 7, 1989. The preferred method of attachment is bycovalent means. Following attachment of a specific binding member, thesurface may be further treated with materials such as serum, proteins,or other blocking agents to minimize non-specific binding. The surfacealso may be scanned either at the site of manufacture or point of use toverify its suitability for assay purposes. The scanning process is notanticipated to alter the specific binding properties of the test piece.

Various other assay formats may be used, including "sandwich"immunoassays and competitive probe assays. For example, the monoclonalantibodies of the present invention can be employed in various assaysystems to determine the presence, if any, of mutant HBV proteins in atest sample. Fragments of these monoclonal antibodies provided also maybe used. For example, in a first assay format, a polyclonal ormonoclonal anti-mutant HBV antibody or fragment thereof, or acombination of these antibodies, which has been coated on a solid phase,is contacted with a test sample which may contain mutant HBV proteins,to form a mixture. This mixture is incubated for a time and underconditions sufficient to form antigen/antibody complexes. Then, anindicator reagent comprising a monoclonal or a polyclonal antibody or afragment thereof, which specifically binds to a mutant HBV region, or acombination of these antibodies, to which a signal generating compoundhas been attached, is contacted with the antigen/antibody complexes toform a second mixture. This second mixture then is incubated for a timeand under conditions sufficient to form antibody/antigen/antibodycomplexes. The presence of mutant HBV antigen present in the test sampleand captured on the solid phase, if any, is determined by detecting themeasurable signal generated by the signal generating compound. Theamount of mutant HBV antigen present in the test sample is proportionalto the signal generated.

Alternatively, a polyclonal or monoclonal anti-mutant HBV antibody orfragment thereof, or a combination of these antibodies which is bound toa solid support, the test sample and an indicator reagent comprising amonoclonal or polyclonal antibody or fragments thereof, whichspecifically binds to mutant HBV antigen, or a combination of theseantibodies to which a signal generating compound is attached, arecontacted to form a mixture. This mixture is incubated for a time andunder conditions sufficient to form antibody/antigen/antibody complexes.The presence, if any, of mutant HBV proteins present in the test sampleand captured on the solid phase is determined by detecting themeasurable signal generated by the signal generating compound. Theamount of mutant HBV proteins present in the test sample is proportionalto the signal generated.

In another alternate assay format, one or a combination of one or moremonoclonal antibodies of the invention can be employed as a competitiveprobe for the detection of antibodies to mutant HBV protein. Forexample, mutant HBV proteins, either alone or in combination with othermutant HBV proteins or non-mutant HBV proteins, can be coated on a solidphase. A test sample suspected of containing antibody to mutant and/ornon-mutant HBV antigen then is incubated with an indicator reagentcomprising a signal generating compound, and at least one monoclonalantibody of the invention for a time and under conditions sufficient toform antigen/antibody complexes of either the test sample and indicatorreagent to the solid phase or the indicator reagent to the solid phase.The reduction in binding of the monoclonal antibody to the solid phasecan be quantitatively measured. A measurable reduction in the signalcompared to the signal generated from a confirmed negative HBV testsample indicates the presence of anti-HBV antibody in the test sample.

In yet another detection method, each of the monoclonal antibodies ofthe present invention can be employed in the detection of mutant HBVantigens in fixed tissue sections, as well as fixed cells byimmunohistochemical analysis.

In addition, these monoclonal antibodies can be bound to matricessimilar to CNBr-activated Sepharose and used for the affinitypurification of specific mutant HBV proteins from cell cultures, orbiological tissues such as blood and liver.

The monoclonal antibodies of the invention can also be used for thegeneration of chimeric antibodies for therapeutic use, or other similarapplications.

The monoclonal antibodies or fragments thereof can be providedindividually to detect mutant HBV antigens. Combinations of themonoclonal antibodies (and fragments thereof) provided herein also maybe used together as components in a mixture or "cocktail" of at leastone anti-mutant HBV antibody of the invention with antibodies to otherHBV regions (either mutant or non-mutant, each having different bindingspecificities. Thus, this cocktail can include the monoclonal antibodiesof the invention which are directed to mutant HBV proteins and othermonoclonal antibodies to other antigenic determinants of the HBV genome.

The polyclonal antibody or fragment thereof which can be used in theassay formats should specifically bind to a specific region of mutantHBV or other mutant HBV proteins used in the assay. The polyclonalantibody used preferably is of mammalian origin; human, goat, rabbit orsheep anti-HCV polyclonal antibody can be used. Most preferably, thepolyclonal antibody is rabbit polyclonal anti-mutant HBV antibody. Thepolyclonal antibodies used in the assays can be used either alone or asa cocktail of polyclonal antibodies. Since the cocktails used in theassay formats are comprised of either monoclonal antibodies orpolyclonal antibodies having different HBV specificity, they would beuseful for diagnosis, evaluation and prognosis of HBV infection, as wellas for studying HBV protein differentiation and specificity.

In another assay format, the presence of antibody and/or antigen tomutant HBV can be detected in a simultaneous assay, as follows. A testsample is simultaneously contacted with a capture reagent of a firstanalyte, wherein said capture reagent comprises a first binding memberspecific for a first analyte attached to a solid phase and a capturereagent for a second analyte, wherein said capture reagent comprises afirst binding member for a second analyte attached to a second solidphase, to thereby form a mixture. This mixture is incubated for a timeand under conditions sufficient to form capture reagent/first analyteand capture reagent/second analyte complexes. These so-formed complexesthen are contacted with an indicator reagent comprising a member of abinding pair specific for the first analyte labelled with a signalgenerating compound and an indicator reagent comprising a member of abinding pair specific for the second analyte labelled with a signalgenerating compound to form a second mixture. This second mixture isincubated for a time and under conditions sufficient to form capturereagent/first analyte/indicator reagent complexes and capturereagent/second analyte/indicator reagent complexes. The presence of oneor more analytes is determined by detecting a signal generated inconnection with the complexes formed on either or both solid phases asan indication of the presence of one or more analytes in the testsample. In this assay format, proteins derived from human expressionsystems may be utilized as well as monoclonal antibodies produced fromthe proteins derived from the mammalian expression systems as disclosedherein. Such assay systems are described in greater detail in pendingU.S. patent application Ser. No. 07/574,821 entitled Simultaneous Assayfor Detecting One Or More Analytes, which corresponds to EP PublicationNo. 0473065, which is incorporated herein by reference.

In yet other assay formats, recombinant proteins may be utilized todetect the presence of anti-mutant HBV in test samples. For example, atest sample is incubated with a solid phase to which at least onerecombinant protein has been attached. These are reacted for a time andunder conditions sufficient to form antigen/antibody complexes.Following incubation, the antigen/antibody complex is detected.Indicator reagents may be used to facilitate detection, depending uponthe assay system chosen. In another assay format, a test sample iscontacted with a solid phase to which a recombinant protein produced asdescribed herein is attached and also is contacted with a monoclonal orpolyclonal antibody specific for the protein, which preferably has beenlabelled with an indicator reagent. After incubation for a time andunder conditions sufficient for antibody/antigen complexes to form, thesolid phase is separated from the free phase, and the label is detectedin either the solid or free phase as an indication of the presence ofHBV antibody. Other assay formats utilizing the proteins of the presentinvention are contemplated. These include contacting a test sample witha solid phase to which at least one recombinant protein produced in themammalian expression system has been attached, incubating the solidphase and test sample for a time and under conditions sufficient to formantigen/antibody complexes, and then contacting the solid phase with alabelled recombinant antigen. Assays such as this and others aredescribed in pending U.S. patent application Ser. No. 07/787,710, whichis incorporated herein by reference.

While the present invention discloses the preference for the use ofsolid phases, it is contemplated that the proteins of the presentinvention can be utilized in non-solid phase assay systems. These assaysystems are known to those skilled in the art, and are considered to bewithin the scope of the present invention.

While the present invention discloses the preference for the use ofsolid phases, it is contemplated that the peptides of the presentinvention can be utilized in non-solid phase assay systems. These assaysystems are known to those skilled in the art, and are considered to bewithin the scope of the present invention.

Materials and Methods

General Techniques

Conventional and well-known techniques and methods in the fields ofmolecular biology, microbiology, recombinant DNA and immunology areemployed in the practice of the invention unless otherwise noted. Suchtechniques are explained and detailed in the literature. See, forexample, T. Maniatis et al., Molecular Cloning: A Laboratory Manual, 2ndedition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); D.N. Glover, ed., DNA Cloning. Volumes I and II (1985); M. J. Gait ed.,Oligonucleotide Synthesis, (1984); B. D. Hames et al., eds., NucleicAcid Hybridization (1984); B. D. Hames et al., eds., Transcription andTranslation, (1984); R. I. Freshney ed., Animal Cell Culture, (1986);Immobilized Cells and Enzymes, IRL Press (1986); B. Perbal, A PracticalGuide to Molecular Cloning, (1984); the series, Methods in Enzymology,Academic Press, Inc., Orlando, Fla.; J. H. Miller et al., eds., GeneTransfer Vectors For Mammalian Cells, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1987); Wu et al., eds., Methods in Enzymology,Vol. 154 and 155; Mayer et al., eds., Immunological Methods In Cell andMolecular Biology, Academic Press, London (1987); Scopes, ProteinPurification: Principles and Practice, 2nd ed., Springer-Verlag, N.Y.;and D. Weir et al., eds., Handbook Of Experimental Immunology, VolumesI-IV (1986).

The reagents and methods of the present invention are made possible bythe provision of a family of closely homologous nucleotide sequencesisolated from a genomic library derived from nucleic acid sequencespresent in the plasma, serum or liver homogenate of a mutant HBVinfected individual. Sera, plasma or liver homogenates from mutant HBVinfected humans contain antibodies which bind to this polypeptide,whereas sera, plasma or liver homogenates from non-infected humans donot contain antibodies to this polypeptide. Finally, whereas the serafrom uninfected individuals do not contain antibodies to thispolypeptide, the antibodies are induced in individuals following acuteHBV infection.

The availability of nucleic acid sequences permits the construction ofDNA probes and polypeptides useful in diagnosing hepatitis due to mutantHBV infections, and in screening blood donors, donated blood, bloodproducts and individuals for infection. For example, from the sequenceit is possible to synthesize DNA oligomers of about eight to tennucleotides, or larger, which re useful as hybridization probes todetect the presence of the viral genome in, for example, sera ofsubjects suspected of harboring the virus, or for screening donatedblood for the presence of the virus. The family of nucleic acidsequences also allows the design and production of mutant HBV specificpolypeptides which are useful as diagnostic reagents for the presence ofantibodies raised during infection with mutant HBV. Antibodies topurified polypeptides derived from the nucleic acid sequences may alsobe used to detect viral antigens in infected individuals and in blood.These nucleic acid sequences also enable the design and production ofpolypeptides which may be used as vaccines against mutant HBV, and alsofor the production of antibodies, which then may be used for protectionof the disease, and/or for therapy of mutant HBV infected individuals.

The sequences and the polypeptides derived from these sequences, as wellas antibodies directed against these polypeptides, also are useful inthe isolation and identification of the mutant HBV etiological agent(s).For example, antibodies directed against mutant HBV epitope contained inpolypeptides derived from the nucleic acid sequences may be used inmethods based upon affinity chromatography to isolate the virus.Alternatively, the antibodies can be used to identify viral particlesisolated by other techniques. The viral antigens and the genomicmaterial within the isolated viral particles then may be furthercharacterized.

The information obtained from further sequencing of the mutant HBVgenome(s), as well as from further characterization of the mutant HBVantigen and characterization of the genome enables the design andsynthesis of additional probes and polypeptides and antibodies which maybe used for diagnosis, prevention and therapy of mutant HBV inducedhepatitis, and for screening for infected blood and blood-relatedproducts.

The availability of probes for mutant HBV, including antigens,antibodies and polynucleotides derived from the genome from which thenucleic acid sequences is derived also allows for the development oftissue culture systems which will be of major use in elucidating thebiology of mutant HBV. Once this is known, it is contemplated that newtreatment regimens may be developed based upon antiviral compounds whichpreferentially inhibit the replication of or infection by mutant HBV.

In the method used to identify and isolate the etiological agent of HBV,a genomic library is created from the nucleic acids present in infectedserum, plasma or liver homogenates from an infected individual,preferably a chimpanzee or human. The library is created in a vectorwhich allows the expression of polypeptides encoded in the nucleic acidsequences. Clones of host cells containing the vector, which hasexpressed an immunologically reactive fragment of a polypeptide of theetiological agent (mutant HBV), are selected by immunological screeningof the expression products of the library with an antibody containingbody component from another individual previously infected with theputative agent. The steps in the immunological screening techniqueinclude interacting the expression products of the cloned nucleic acidsequences containing vectors with the antibody containing body componentof a second infected individual, and detecting the formation ofantigen-antibody complexes between the expression product(s) andantibodies of the second infected individual. The isolated clones arescreened further immunologically by interacting their expressionproducts with the antibody containing body component of otherindividuals infected with the putative agent and detecting the formationof antigen-antibody complexes with antibodies from the infectedindividuals, and the nucleic acid sequences containing vectors whichencode polypeptides which react immunologically with antibodies frominfected individuals and individuals suspected of being infected theagent, but not with control individuals, are isolated. The infectedindividuals used for the construction of the nucleic acid sequencelibrary, and for the immunological screening need not be of the samespecies. The nucleic acid sequences isolated as a result of this method,and their expression products, and antibodies directed against theexpression products, are useful in characterizing and/or capturing theetiological agent. This method is taught in EP patent applicationPublication No. 0 318 216, which is incorporated herein by reference.

Preparation of the Nucleic Acid Sequences

Pooled or individual serum, plasma or liver homogenates from anindividual meeting the criteria and within the parameters set forthbelow with acute or chronic mutant HBV infection is used to isolateviral particles. Nucleic acids isolated from these particles is used asthe template in the construction of a genomic library to the viralgenome. The procedures used for isolation of mutant HBV particles andfor constructing the genomic library in lambda-gt11 or similar systemsknown in the art is discussed hereinbelow. Lambda-gt11 is a vector thathas been developed specifically to express inserted cDNAs as fusionpolypeptides with beta-galactosidase and to screen large numbers ofrecombinant phage with specific antisera raised against a definedantigen. The lambda-gt11 cDNA library generated from a cDNA poolcontaining cDNA is screened for encoded epitopes that can bindspecifically with sera derived from individuals who previously hadexperienced hepatitis due to mutant HBV. See V. Hunyh et al., in D.Glover, ed, DNA Cloning Techniques: A Practical Approach, IRLPress,Oxford, England, pp. 49-78 (1985). Approximately 10⁶ -10⁷ phagesare screened, from which positive phages are identified, purified, andthen tested for specificity of binding to sera from differentindividuals previously infected with the mutant HBV agent. Phages whichselectively bind sera, plasma from patients meeting the criteriadescribed hereinbelow and not in patients who did not meet thesedescribed criteria, are preferred for further study.

By utilizing the technique of isolating overlapping nucleic acidsequences, clones containing additional upstream and downstream mutantHBV sequences are obtained. The isolation of these clones is describedhereinbelow.

Analysis of the nucleotide sequences of the mutant HBV nucleic acidsequences encoded within the isolated clones is performed to determinewhether the composite sequence contains one long continuous ORF. Thesequences (and their complements) retrieved from the mutant HBV libraryof sequences are provided herein, and the sequences or any portionthereof, can be prepared using synthetic methods or by a combination ofsynthetic methods with retrieval of partial sequences using methodssimilar to those described herein. This description thus provides onemethod by which genomic sequences corresponding to the entire mutant HBVgenome may be isolated. Other methods for isolating these sequences,however, will be obvious to those skilled in the art and are consideredto be within the scope of the present invention.

Strains replicated from the mutant HBV nucleic acid sequence librarywill be deposited at the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852, under the terms of the BudapestTreaty and will be maintained for a period of thirty (30) years from thedate of deposit, or for five (5) years after the last request for thedeposit, or for the enforceable period of the U.S. patent, whichever islonger. The deposits and any other deposited material described hereinare provided for convenience only, and are not required to practice thepresent invention in view of the teachings provided herein.

Preparation of Viral Polypeptides and Fragments

The availability of nucleic acid sequences permits the construction ofexpression vectors encoding antigenically active region of thepolypeptide encoded in either strand. The antigenically active region isderived from envelope (coat) antigen. Fragments encoding the desiredpolypeptides are derived from the genomic clones using conventionalrestriction digestion or by synthetic methods, and are ligated intovectors which may, for example, contain portions of fusion sequencessuch as beta-galactosidase (B-gal) or superoxide dismutase (SOD) orCMP-KDO synthetase (CKS). Methods and vectors which are useful for theproduction of polypeptides which contain fusion sequences of SOD aredescribed in EPO 0196056, published Oct. 1, 1986, and those of CKS aredescribed in EPO Publication No. 0331961, published Sep. 13, 1989, whichare incorporated herein by reference. Any desired portion of the nucleicacid sequence containing an open reading frame, in either sense strand,can be obtained as a recombinant protein, such as a mature or fusionprotein; alternatively, a polypeptide encoded in the mutant HBV genomecan be provided by chemical synthesis.

The nucleic acid sequence encoding the desired polypeptide, whether infused or mature form, and whether or not containing a signal sequence topermit secretion, may be ligated into expression vectors suitable forany convenient host. Both eucaryotic and prokaryotic host systems areused in the art to form recombinant proteins, and some of these arelisted herein. The polypeptide then is isolated from lysed cells or fromthe culture medium and purified to the extent needed for its intendeduse. Purification can be performed by techniques known in the art, andinclude salt fractionation, chromatography on ion exchange resins,affinity chromatography, centrifugation, among others. Such polypeptidesmay be used as diagnostic reagents, or for passive immunotherapy. Inaddition, antibodies to these polypeptides are useful for isolating andidentifying mutant HBV particles. The mutant HBV antigens also may beisolated from mutant HBV virions. These virions can be grown in mutantHBV infected cells in tissue culture, or in an infected individual.

Preparation of Antigenic Polypeptides and Conjugation With Solid Phase

An antigenic region or fragment of a polypeptide generally is relativelysmall, usually about 8 to 10 amino acids or less in length. Fragments ofas few as 2-5 amino acids may characterize an antigenic region. Thesesegments may correspond to regions of the mutant HBV antigen. By usingthe mutant HBV genomic sequences as a basis, nucleic acid sequencesencoding short segments of mutant HBV polypeptides can be expressedrecombinantly either as fusion proteins or as isolated polypeptides.These short amino acid sequences also can be obtained by chemicalsynthesis. The small chemically synthesized polypeptides may be linkedto a suitable carrier molecule when the synthesized polypeptide providedis correctly configured to provide the correct epitope but too small tobe antigenic. Linking methods are known in the art and include but arenot limited to using N-succinimidyl-3-(2-pyrdylthio)propionate (SPDP)and succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).Polypeptides lacking sulfhydryl groups can be modified by adding acysteine residue. These reagents create a disulfide linkage betweenthemselves and peptide cysteine residues on one protein and an amidelinkage through the epsilon-amino on a lysine, or other free amino groupin the other. A variety of such disulfidetamide-forming agents areknown. Other bifunctional coupling agents form a thioester rather than adisulfide linkage. Many of these thio-ether-forming agents arecommercially available and are known to those of ordinary skill in theart. The carboxyl groups can be activated by combining them withsuccinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Anycarrier which does not itself induce the production of antibodiesharmful to the host can be used. Suitable carriers include proteins,polysaccharides such as latex functionalized sepharose, agarose,cellulose, cellulose beads, polymeric amino acids such as polyglutamicacid, polylysine, amino acid copolymers and inactive virus particles,among others. Examples of protein substrates include serum albumins,keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin,ovalbumin, tetanus toxoid, and yet other proteins known to those skilledin the art.

Preparation of Hybrid Particle Immunogens Containing HBV Epitopes

The immunogenicity of mutant HBV epitope(s) also may be enhanced bypreparing them in mammalian or yeast systems fused with or assembledwith particle-forming proteins such as those associated with HBV surfaceantigen. Constructs wherein the mutant HBV epitope is linked directly tothe particle-forming protein coding sequences produce hybrids which areimmunogenic with respect to the mutant HBV epitope. In addition, all ofthe vectors prepared include epitopes specific for mutant HBV, havingvarying degrees of immunogenicity. Particles constructed from particleforming protein which include mutant HBV sequences are immunogenic withrespect to mutant HBV.

Hepatitis B surface antigen has been determined to be formed andassembled into particles in S. cerevisiae and mammalian cells; theformation of these particles has been reported to enhance theimmunogenicity of the monomer subunit. P. Valenzuela et al., Nature298:334 (1982); P. Valenzuela et al., in I. Millman et al., eds.,Hepatitis B, Plenum Press, pp. 225-236 (1984). The constructs mayinclude immunodominant epitopes of HBsAg. Such constructs have beenreported expressible in yeast, and hybrids including heterologous viralsequences for yeast expression have been disclosed. See, for example,EPO 174,444 and EPO 174,261. These constructs also have been reportedcapable of being expressed in mammalian cells such as Chinese hamsterovary (CHO) cells. Michelle et al., International Symposium on ViralHepatitis, 1984. In HBV, portions of the particle-forming protein codingsequence may be replaced with codons encoding an HBV epitope. In thisreplacement, regions that are not required to mediate the aggregation ofthe units to form immunogenic particles in yeast or mammals can bedeleted, thus eliminating additional HBV antigenic sites fromcompetition with the HBV epitope.

Vaccine Preparation

Vaccines may be prepared from one or more immunogenic polypeptidesderived from mutant HBV nucleic acid sequences or from the mutant HBVgenome to which they correspond. Vaccines may comprise recombinantpolypeptides containing epitope(s) of mutant HBV. These polypeptides maybe expressed in bacteria, yeast or mammalian cells, or alternatively maybe isolated from viral preparations. It also is anticipated that variousstructural proteins may contain epitopes of mutant HBV which give riseto protective anti-mutant HBV antibodies. Thus, polypeptides containingat least one epitope of mutant HBV may be used, either singly or incombinations, in mutant HBV vaccines. It also is contemplated thatnonstructural proteins as well as structural proteins may provideprotection against viral pathogenicity, even if they do not cause theproduction of neutralizing antibodies.

Considering the above, multivalent vaccines against mutant HBV maycomprise proteins which include the two amino acid insertion (N-T) atposition 122. These vaccines may be comprised of, for example,recombinant mutant HBV polypeptides and/or polypeptides isolated fromthe virions. Additionally, it may be possible to use inactivated mutantHBV in vaccines. Such inactivation may be by preparation of virallysates, or by other means known in the art to cause inactivation ofhepatitis-like viruses, for example, treatment with organic solvents ordetergents, or treatment with formalin. Attenuated mutant HBV strainpreparation also is disclosed in the present invention. It iscontemplated that some of the proteins in mutant HBV may cross-reactwith other known viruses, and thus that shared epitopes may existbetween mutant HBV and other viruses which would then give rise toprotective antibodies against one or more of the disorders caused bythese pathogenic agents. It is contemplated that it may be possible todesign multiple purpose vaccines based upon this belief.

The preparation of vaccines which contain at least one immunogenicpeptide as an active ingredient is known to one skilled in the art.Typically, such vaccines are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in orsuspension in liquid prior to injection also may be prepared. Thepreparation may be emulsified. or the protein may be encapsulated inliposomes. The active immunogenic ingredients often are mixed withpharmacologically acceptable excipients which are compatible with theactive ingredient. Suitable excipients include but are not limited towater, saline, dextrose, glycerol, ethanol and the like; combinations ofthese excipients in various amounts also may be used. The vaccine alsomay contain small amounts of auxiliary substances such as wetting oremulsifying reagents, pH buffering agents, and/or adjuvants whichenhance the effectiveness of the vaccine. For example, such adjuvantscan include aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine(CGP 19835A, also referred to as MTP-PE), and RIBI (MPL+TDM+CWS) in a 2%squalene/Tween-80® emulsion. The effectiveness of an adjuvant may bedetermined by measuring the amount of antibodies directed against animmunogenic polypeptide containing an HBV antigenic sequence resultingfrom administration of this polypeptide in vaccines which also arecomprised of the various adjuvants.

The vaccines usually are administered by intraveneous or intramuscularinjection. Additional formulations which are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude but are not limited to polyalkylene glycols or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10%, preferably, about 1%to about 2%. Oral formulation include such normally employed excipientsas, for example pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonateand the like. These compositions may take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain about 10% to about 95% of active ingredient,preferably about 25% to about 70%.

The proteins used in the vaccine may be formulated into the vaccine asneutral or salt forms. Pharmaceutically acceptable salts such as acidaddition salts (formed with free amino groups of the peptide) and whichare formed with inorganic acids such as hydrochloric or phosphoricacids, or such organic acids such as acetic, oxalic, tartaric, maleic,and others known to those skilled in the art. Salts formed with the freecarboxyl groups also may be derived from inorganic bases such as sodium,potassium, ammonium, calcium or ferric hydroxides and the like, and suchorganic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine procaine, and others known to those skilled in theart.

Vaccines are administered in a way compatible with the dosageformulation, and in such amounts as will be prophylactically and/ortherapeutically effective. The quantity to be administered generally isin the range of about 5 micrograms to about 250 micrograms of antigenper dose, and depends upon the subject to be dosed, the capacity of thesubject's immune system to synthesize antibodies, and the degree ofprotection sought. Precise amounts of active ingredient required to beadministered also may depend upon the judgment of the practitioner andmay be unique to each subject. The vaccine may be given in a single ormultiple dose schedule. A multiple dose is one in which a primary courseof vaccination may be with one to ten separate doses, followed by otherdoses given at subsequent time intervals required to maintain and/or toreenforce the immune response, for example, at one to four months for asecond dose, and if required by the individual, a subsequent dose(s)after several months. The dosage regimen also will be determined, atleast in part, by the need of the individual, and be dependent upon thepractitioner's judgment It is contemplated that the vaccine containingthe immunogenic mutant HBV antigen(s) may be administered in conjunctionwith other immunoregulatory agents, for example, with immune globulins.

Preparation of Antibodies Against Mutant HBV Epitopes

The immunogenic peptides prepared as described herein are used toproduce antibodies, either polyclonal or monoclonal. When preparedpolyclonal antibodies, a selected mammal (for example, a mouse, rabbit,goat, horse and the like) is immunized with an immunogenic polypeptidebearing at least one mutant HBV epitope. Serum from the immunized animalis collected after an appropriate incubation period and treatedaccording to known procedures. If serum containing polyclonal antibodiesto an epitope of mutant HBV contains antibodies to other antigens, thepolyclonal antibodies can be purified by, for example, immunoaffinitychromatography. Techniques for producing and processing polyclonalantibodies are known in the art and are described in, among others,Mayer and Walker, eds., Immunochemical Methods In Cell and MolecularBiology, Academic Press, London (1987). Polyclonal antibodies also canbe isolated. Polyclonal antibodies may be obtained from a mammalpreviously infected with HBV. An example of a method for purifyingantibodies to mutant HBV epitopes from serum of an individual infectedwith mutant HBV using affinity chromatography is provided herein.

Monoclonal antibodies directed against mutant HBV epitopes also canproduced by one skilled in the art. The general methodology forproducing such antibodies is well-known and has been described in, forexample, Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.G. R. Hurrel, ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press Inc., Boco Ratan, Fla. (1982), as well as thattaught by L. T. Mimms et al., Virology 176:604-619 (1990). Immortalantibody-producing cell lines can be created by cell fusion, and also byother techniques such as direct transformation of B lymphocytes withoncogenic DNA, or transfection with Epstein-Barr virus. See also, M.Schreier et al., Hybridoma Techniques. Scopes (1980) ProteinPurification, Principles and Practice, 2nd Edition, Springer-Verlag,N.Y. (1984); Hammerling et al., Monoclonal Antibodies and T-CellHybridomas (1981); Kennet et al., Monoclonal Antibodies (1980). Examplesof uses and techniques of monoclonal antibodies for HCV are disclosed inU.S. patent application Ser. Nos. 748,292; 748,563; 610,175, 648,473;648,477; and 648,475.

Monoclonal and polyclonal antibodies thus developed, directed againstmutant HBV epitopes, are useful in diagnostic and prognosticapplications, and also, those which are neutralizing are useful inpassive immunotherapy. Monoclonal antibodies especially can be used toproduce anti-idiotype antibodies. These anti-idiotype antibodies areimmunoglobulins which carry an "internal image" of the antigen of theinfectious agent against which protection is desired. See, for example,A. Nisonoff et al., Clin. Immunol. Immunopath. 21:397-406 (1981), andDreesman et al., J. Infect. Dis. 151:761 (1985). Techniques for raisingsuch idiotype antibodies are known in the art and exemplified, forexample, in Grych et al., Nature 316:74 (1985); MacNamara et al.,Science 226:1325 (1984); and Uytdehaag et al., J. Immunol. 134:1225(1985). These anti-idiotypic antibodies also may be useful for treatmentof HBV infection, as well as for elucidation of the immunogenic regionsof HBV antigens.

Diagnostic Oligonucleotide Probes and Kits

Using determined portions of the isolated mutant HBV nucleic acidsequences as a basis, oligomers of approximately 8 nucleotides or morecan be prepared, either by excision or synthetically, which hybridizewith the mutant HBV genome and are useful in identification of the viralagent(s), further characterization of the viral genome, as well as indetection of the virus(es) in diseased individuals. The natural orderived probes for mutant HBV polynucleotides are a length which allowsthe detection of unique viral sequences by hybridization. While 6 to 8nucleotides may be a workable length, sequences of 10 to 12 nucleotidesare preferred, and those of about 20 nucleotides may be most preferred.These sequences preferably will derive from regions which lackheterogeneity. These probes can be prepared using routine, standardmethods including automated oligonucleotide synthetic methods. Acomplement of any unique portion of the mutant HBV genome will besatisfactory. Complete complementarity is desirable for use as probes,although it may be unnecessary as the length of the fragment isincreased.

When used as diagnostic reagents, the biological test sample to beanalyzed, such as blood or serum may be treated such as to extract thenucleic acids contained therein. The resulting nucleic acid from thesample may be subjected to gel electrophoresis or other size separationtechniques; or, the nucleic acid sample may be dot-blotted without sizeseparation. The probes then are labelled. Suitable labels are methodsfor attaching labels to probes are known in the art, and include but arenot limited to radioactive labels incorporated by nick translation orkinasing, biotin, fluorescent and chemiluminescent probes. Examples ofmany of these labels are disclosed herein. The nucleic acids extractedfrom the sample then are treated with the labelled probe underhybridization conditions of suitable stringencies.

The probes can be made completely complementary to the mutant HBVgenome. Therefore, usually high stringency conditions are desirable inorder to prevent false positives. However, conditions of high stringencyshould be used only if the probes are complementary to regions of themutant HBV genome which lack heterogeneity. The stringency ofhybridization is determined by a number of factors during the washingprocedure, including temperature, ionic strength, length of time andconcentration of formamide. See, for example, T. Maniatis (supra).Hybridization can be carried out by a number of various techniques,including, for example, by Ligase Chain Reaction (LCR), Polymerase ChainReaction (PCR). These techniques are described herein.

It is contemplated that the mutant HBV genome sequences may be presentin serum of infected individuals at relatively low levels, for example,approximately 10² -10³ sequences per ml. This level may require thatamplification techniques be used in hybridization assays, such as theLigase Chain Reaction or the Polymerase Chain Reaction. Such techniquesare known in the art. For example, the "Bio-Bridge" system uses terminaldeoxynucleotide transferase to add unmodified 3'-poly-dT-tails to anucleic acid probe (Enzo Biochem. Corp.). The poly dt-tailed probe ishybridized to the target nucleotide sequence, and then to abiotin-modified poly-A. Also, in EP 124221 there is described a DNAhybridization assay wherein the analyte is annealed to a single-strandedDNA probe that is complementary to an enzyme-labelled oligonucleotide,and the resulting tailed duplex is hybridized to an enzyme-labeledoligonucleotide. EP 204510 describes a DNA hybridization assay in whichanalyte DNA is contacted with a probe that has a tail, such as apoly-dt-tail, an amplifier strand that has a sequence that hybridizes toto the tail of the probe, such as a poly-A sequence, and which iscapable of binding a plurality of labelled strands. The technique firstmay involve amplification of the target HBV sequences in sera toapproximately 10⁶ sequences/ml. This may be accomplished by followingthe methods described by Saiki et al., Nature 324:163 (1986). Theamplified sequence(s) then may be detected using a hybridization assaysuch as those known in the art. The probes can be packaged in diagnostickits which include the probe nucleic acid sequence which sequence may belabelled; alternatively, the probe may be unlabelled and the ingredientsfor labelling could be included with the kit. The kit also may containother suitably packaged reagents and materials needed or desirable forthe particular hybridization protocol, for example, standards as well asinstructions for performing the assay.

Immunoassay and Diagnostic Kits

Both the polypeptides which react immunologically with serum containingmutant HBV antibodies and composites thereof, and the antibodies raisedagainst the mutant HBV specific epitopes in these polypeptides areuseful in immunoassays to detect the presence of mutant HBV antibodies,or the presence of the virus and/or viral antigens in biological testsamples. The design of these immunoassays is subject to variation, and avariety of these are known in the art; a variety of these have beendescribed herein. The immunoassay may utilize one viral antigen, such asa polypeptide derived from any clone-containing mutant HBV nucleic acidsequence, or from the composite nucleic acid sequences derived from themutant HBV nucleic acid sequences in these clones, or from the mutantHBV genome from which the nucleic acid sequences in these clones isderived. Or, the immunoassay may use of combination of viral antigensderived from these sources. It may use, for example, a monoclonalantibody directed against the same viral antigen, or polyclonalantibodies directed against different viral antigens. Assays can includebut are not limited to those based on competition, direct reaction orsandwich-type assays. Assays may use solid phases or may be performed byimmunoprecipitation or any other methods which do not utilize solidphases. Examples of assays which utilize labels as the signal generatingcompound and those labels are described herein. Signals also may beamplified by using biotin and avidin, enzyme labels or biotinanti-biotin systems, such as that described in pending U.S. patentapplication Serial Nos. 608,849; 070,647; 418,981; and 687,785.Recombinant polypeptides which include epitopes from immunodominantregions of mutant HBV may be useful for the detection of viralantibodies in biological test samples of infected individuals. It alsois contemplated that antibodies may be useful in discriminating acutefrom non-acute infections. Kits suitable for imrnmunodiagnosis andcontaining the appropriate reagents are constructed by packaging theappropriate materials, including the polypeptides of the inventioncontaining mutant HBV epitopes or antibodies directed against mutant HBVepitopes in suitable containers, along with the remaining reagents andmaterials required for the conduct of the assay, as well as suitableassay instructions.

Further Characterization of the HBV Genome, Virions, and Viral AntigensUsing Probes

The mutant HBV nucleic acid sequences may be used to gain furtherinformation on the sequence of the mutant HBV genome, and foridentification and isolation of mutant HBV. This information, in turn,can lead to additional polynucleotide probes, polypeptides derived fromthe HBV genome, and antibodies directed against mutant HBV epitopeswhich would be useful for the diagnosis and/or treatment of mutant HBVhepatitis.

The nucleic acid sequence information is useful for the design of probesfor the isolation of additional nucleic acid sequences which are derivedfrom the envelope region of the mutant HBV genome. For example, labelledprobes containing a sequence of 8 or more nucleotides, and preferably 20or more nucleotides, which are derived from regions close to the5'-termini or 3'-termini of mutant HBV nucleic acid sequences may beused to isolate overlapping nucleic acid sequences from mutant HBVgenomic libraries. These sequences which overlap the mutant HBV nucleicacid sequences, but which also contain sequences derived from regions ofthe genome from which the above-mentioned mutant HBV nucleic acidsequence are not derived, may then be used to synthesize probes foridentification of other overlapping fragments which do not necessarilyoverlap the nucleic acid sequences in the clones. Unless the mutant HBVgenome is segmented and the segments lack common sequences, it ispossible to sequence the entire viral genome(s) utilizing the techniqueof isolation of overlapping nucleic acid sequences derived from theviral genome(s). Although it is unlikely, if the genome is a segmentedgenome which lacks common sequences, the sequence of the genome can bedetermined serologically by screening lambda-gt11 mutant HBV genomiclibraries, sequencing mutant HBV genomic isolates, and using theisolated mutant HBV nucleic acid sequences to isolate overlappingfragments, using the techniques described for the isolation andsequencing of clones. Characterization of the genomic segmentsalternatively could be from the viral genome(s) isolated from purifiedmutant HBV particles. Methods for purifying mutant HBV particles and fordetecting them during the purification procedure are described herein.Procedures for isolating polynucleotide genomes from viral particles arewell-known in the art. The isolated genomic segments then could becloned and sequenced. Thus, it is possible to clone and sequence themutant HBV genome(s) irrespective of its nature.

Methods for constructing mutant HBV genomic libraries are known in theart, and vectors useful for this purpose are known in the art. Thesevectors include lambda-gt11, lambda-gt10, and others. The mutant HBVderived nucleic acid sequence detected by the probes derived from themutant HBV genomic libraries, may be isolated from the clone bydigestion of the isolated polynucleotide with the appropriaterestriction enzyme(s), and sequenced.

The sequence information derived from these overlapping mutant HBVnucleic acid sequences is useful for determining areas of homology andheterogeneity within the viral genome(s), which could indicate thepresence of different strains of the genome and or of populations ofdefective particles. It is also useful for the design of hybridizationprobes to detect mutant HBV or mutant HBV antigens or mutant HBV nucleicacids in biological samples, and during the isolation of mutant HBV,utilizing the techniques described herein. The overlapping nucleic acidsequences may be used to create expression vectors for polypeptidesderived from the mutant HBV genome(s). Encoded within the family ofnucleic acid sequences are antigen(s) containing epitopes which arecontemplated to be unique to mutant HBV, i.e., antibodies directedagainst these antigens are absent from individuals infected with HAV,HCV, and HEV, and with the genomic sequences in Genebank arecontemplated to indicate that minimal homology exists between thesenucleic acid sequences and the polynucleotide sequences of thosesources. Thus, antibodies directed against the antigens encoded with themutant HBV nucleic acid sequences may be used to identify the mutant HBVparticle isolated from infected individuals. In addition, they also areuseful for the isolation of the mutant HBV agent(s).

Mutant HBV particles may be isolated from the sera of infectedindividuals or from cell cultures by any of the methods known in theart, including, for example, techniques based on size discriminationsuch as sedimentation or exclusion methods, or techniques based ondensity such as ultracentrifugation in density gradients, orprecipitation with agents such as polyethylene glycol (PEG), orchromatography on a variety of materials such as anionic or cationicexchange materials, and materials which bind due to hydrophobicinteractions, as well as affinity columns. During the isolationprocedure the presence of mutant HBV may be detected by hybridizationanalysis of the extracted genome, using probes derived from mutant HBVnucleic acid sequences or by immunoassay which utilize as probesantibodies directed against mutant HBV antigens encoded within thefamily of mutant HBV nucleic acid sequences. The antibodies may bepolyclonal or monoclonal, and it may be desirable to purify theantibodies before their use in the immunoassay. Such antibodies directedagainst mutant HBV antigens which are affixed to solid phases are usefulfor the isolation of mutant HBV by immunoaffinity chromatography.Methods for immunoaffmity chromatography are known in the art, andinclude methods for affixing antibodies to solid phases so that theyretain their immunoselective activity. These methods include adsorption,and covalent binding. Spacer groups may be included in the bifunctionalcoupling agents such that the antigen binding site of the antibodyremains accessible.

During the purification procedure the presence of mutant HBV may bedetected and/or verified by nucleic acid hybridization, utilizing asprobes polynucleotides derived from a family of HBV genomic sequences,as well as from overlapping mutant HBV nucleic acid sequences. Fractionsare treated under conditions which would cause the disruption of viralparticles, such as by use of detergents in the presence of chelatingagents, and the presence of viral nucleic acid determined byhybridization techniques. Further confirmation that the isolatedparticles are the agents which induce mutant HBV infection may beobtained by infecting an individual which is preferably a chimpanzeewith the isolated virus particles, followed by a determination ofwhether the symptoms of mutant HBV hepatitis, as described herein,result from the infection.

Determination of polypeptides containing conserved sequences may beuseful for selecting probes which bind the mutant HBV genome, thusallowing its isolation. In addition, conserved sequences in conjunctionwith those derived from the mutant HBV nucleic acid sequences, may beused to design primers for use in systems which amplify genomicsequences. Further, the structure of mutant HBV also may be determinedand its components isolated. The morphology and size may be determinedby electron microscopy, for example. The identification and localizationof specific viral polypeptide antigens such as envelope (coat) antigens,or internal antigens such as nucleic acid binding proteins or coreantigens, and polynucleotide polymerase(s) also may be determined byascertaining whether the antigens are present in major or minor viralcomponents, as well as by utilizing antibodies directed against thespecific antigens encoded within isolated nucleic acid sequences asprobes. This information may be useful for diagnostic and therapeuticapplications. For example, it may be preferable to include an exteriorantigen in a vaccine preparation, or perhaps multivalent vaccines may becomprised of a polypeptide derived from the genome encoding a structuralprotein as well as a polypeptide from another portion of the genome,such as a nonstructural polypeptide.

Cell Culture Systems and Animal Model Systems for Mutant HBV Replication

Generally, suitable cells or cell lines for culturing mutant HBV mayinclude the following: monkey kidney cells such as MK2 and VERO, porcinekidney cell lines such as PS, baby hamster kidney cell lines such asBHK, murine macrophage cell lines such as P388D1, MK1 and Mm1, humanmacrophage cell lines such as U-937, human peripheral blood leukocytes,human adherent monocytes, hepatocytes or hepatocytic cell lines such asHUH7 and HepG2, embryos or embryonic cell such as chick embryofibroblasts or cell lines derived from invertebrates, preferably frominsects such as drosophia cell lines or more preferably from arthropodssuch as mosquito cell lines or tick cell lines. It also is possible thatprimary hepatocytes can be cultured and then infected with mutant HBV.Alternatively, the hepatocyte cultures could be derived from the liversof infected individuals (human or chimpanzee). That latter case is anexample of a cell line which is infected in vivo being passaged invitro. In addition, various immortalization methods can be used toobtain cell lines derived from hepatocyte cultures. For example, primaryliver cultures (before and after enrichment of the hepatocytepopulation) may be fused to a variety of cells to maintain stability.Also, cultures may be infected with transforming viruses, or transfectedwith transforming genes in order to create permanent or semipermanentcell lines. In addition, cells in liver cultures may be fused toestablished cell lines such as PehG2. Methods for cell fusion arewell-known to the routineer, and include the use of fusion agents suchas PEG, Sendai Virus and Epstein-Barr Virus, among others.

It is contemplated that mutant HBV infection of cell lines may beaccomplished by techniques such as incubating the cells with viralpreparations under conditions which allow viral entry into the cell. Italso may be possible to obtain viral production by transfecting thecells with isolated viral polynucleotides. Methods for transfectingtissue culture cells are known in the art and include but are notlimited to techniques which use electroporation and precipitation withDEAE-Dextran or calcium phosphate. Transfection with cloned mutant HBVgenomic DNA should result in viral replication and the in vitropropagation of the virus. In addition to cultured cells, animal modelsystems may be used for viral replication.

Screening for Anti-Viral Agents For Mutant HBV

The availability of cell culture and animal model systems for mutant HBValso renders screening for anti-viral agents which inhibit mutant HBVreplication possible, and particularly for those agents whichpreferentially allow cell growth and multiplication while inhibitingviral replication. These screening methods are known in the art.Generally, the anti-viral agents are tested at a variety ofconcentrations, for their effect on preventing viral replication in cellculture systems which support viral replication, and then for aninhibition of infectivity or of viral pathogenicity, and a low level oftoxicity, in an animal model system. The methods and compositionprovided herein for detecting mutant HBV antigens and mutant HBVpolynucleotides are useful for screening of anti-viral agents becausethey provide an alternative, and perhaps a more sensitive means, fordetecting the agent's effect on viral replication than the cell plaqueassay or ID₅₀ assay. For example, the mutant HBV polynucleotide probesdescribed herein may be used to quantitate the amount of viral nucleicacid produced in a cell culture. This could be performed byhybridization or competition hybridization of the infected cell nucleicacids with a labelled mutant HBV polynucleotide probe. Also, anti-mutantHBV antibodies may be used to identify and quantitate mutant HBVantigen(s) in the cell culture utilizing the immunoassays describedherein. Also, since it may be desirable to quantitate mutant HBVantigens in the infected cell culture by a competition assay, thepolypeptides encoded within the mutant HBV nucleic acid sequencesdescribed herein are useful for these assays. Generally, a recombinantmutant HBV polypeptide derived from the mutant HBV genomic DNA would belabelled, and the inhibition of binding of this labelled polypeptide toa mutant HBV polypeptide due to the antigen produced in the cell culturesystem would be monitored. These methods are especially useful in caseswhere the mutant HBV may be able to replicate in a cell lines withoutcausing cell death.

Preparation of Attenuated Strains of Mutant HBV

It maybe possible to isolate attenuated strains of mutant HBV byutilizing the tissue culture systems and/or animal models systemsprovided herein. These attenuated strains would be useful for vaccines,or for the isolation of viral antigens. Attenuated strains areisolatable after multiple passages in cell culture and/or an animalmodel. Detection of an attenuated strain in an infected cell orindividual is achievable by following methods known in the art and couldinclude the use of antibodies to one or more epitopes encoded in mutantHBV as a probe or the use of a polynucleotide containing an mutant HBVsequence of at least about 8 nucleotides in length as a probe. Also oralternatively, an attenuated strain may be constructed utilizing thegenomic information of mutant HBV provided herein, and utilizingrecombinant techniques. Usually an attempt is made to delete a region ofthe genome encoding a polypeptide related to pathogenicity but not toviral replication. The genomic construction would allow the expressionof an epitope which gives rise to neutralizing antibodies for mutantHBV. The altered genome then could be used to transform cells whichallow mutant HBV replication, and the cells grown under conditions toallow viral replication. Attenuated mutant HBV strains are useful notonly for vaccine purposes, but also as sources for the commercialproduction of viral antigens, since the processing of these viruseswould require less stringent protection measures for the employeesinvolved in viral production and/or the production of viral products.

Hosts and Expression Control Sequences

Although the following are known in the art, included herein are generaltechniques used in extracting the genome from a virus, preparing andprobing a genomic library, sequencing clones, constructing expressionvectors, transforming cells, performing immunological assays, and forgrowing cell in culture.

Both procaryotic and eukaryotic host cells may be used for expression ofdesired coding sequences when appropriate control sequences which arecompatible with the designated host are used. Among prokaryotic hosts,E. coli is most frequently used. Expression control sequences forprokaryotics include promoters, optionally containing operator portions,and ribosome binding sites. Transfer vectors compatible with prokaryotichosts are commonly derived from the plasmid pBR322 which containsoperons conferring ampicillin and tetracycline resistance, and thevarious pUC vectors, which also contain sequences conferring antibioticresistance markers. These markers may be used to obtain successfultransformants by selection. Commonly used prokaryotic control sequencesinclude the beta-lactamase (penicillinase), lactose promoter system(Chang et al., Nature 198:1056 1977!) the tryptophan promoter system(reported by Goeddel et al., Nucleic Acid Res 8:4057 1980!) and thelambda-derived Pl promoter and N gene ribosome binding site (Shimatakeet al., Nature 292:128 1981!) and the hybrid Tac promoter (De Boer etal., Proc. Natl. Acad. Sci. USA 292:128 1983!) derived from sequences ofthe trp and lac UV5 promoters. The foregoing systems are particularlycompatible with E. coli; however, other prokaryotic hosts such asstrains of Bacillus or Pseudomonas may be used if desired, withcorresponding control sequences.

Eukaryotic hosts include yeast and mammalian cells in culture systems.Saccharomyces cerevisiae and Saccharomyces carlsbergensis are the mostcommonly used yeast hosts, and are convenient fungal hosts. Yeastcompatible vectors carry markers which permit selection of successfultransformants by conferring protrophy to auxotrophic mutants orresistance to heavy metals on wild-type strains. Yeast compatiblevectors may employ the 2 micron origin of replication (as described byBroach et al., Meth. Enz. 101:307 1983!), the combination of CEN3 andARS1 or other means for assuring replication, such as sequences whichwill result in incorporation of an appropriate fragment into the hostcell genome. Control sequences for yeast vectors are known in the artand include promoters for the synthesis of glycolytic enzymes, includingthe promoter for 3 phosphophycerate kinase. See, for example, Hess etal., J. Adv. Enzyme Reg. 7: 149 (1968), Holland et al., Biochemistry17:4900 (1978) and Hitzeman J. Biol. Chem. 255:2073 (1980). Terminatorsalso may be included, such as those derived from the enolase gene asreported by Holland, J. Biol. Chem. 256:1385 (1981). It is contemplatedthat particularly useful control systems are those which comprise theglyceraldehyde-3 phosphate dehydrogenase (GAPDH) promoter or alcoholdehydrogenase (ADH) regulatable promoter, terminators also derived fromGAPDH, and if secretion is desired, leader sequences from yeast alphafactor. In addition, the transcriptional regulatory region and thetranscriptional initiation region which are operably linked may be suchthat they are not naturally associated in the wild-type organism.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines which are available fromthe American Type Culture Collection. These include HeLa cells, Chinesehamster ovary (CHO) cells, baby hamster kidney (BHK) cells, and others.Suitable promoters for mammalian cells also are known in the art andinclude viral promoters such as that from Simian Virus 40 (SV40), Roussarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus (BPV),cytomegalovirus (CMV). Mammalian cells also may require terminatorsequences and poly A addition sequences; enhancer sequences whichincrease expression also may be included, and sequences which causeamplification of the gene also may be desirable. These sequences areknown in the art. Vectors suitable for replication in mammalian cellsmay include viral replicons, or sequences which insure integration ofthe appropriate sequences encoding HBU mutant epitopes into the hostgenome. An example of a mammalian expression system for HCV is describedin U.S. patent application Ser. No. 07/830,024, filed Jan. 31, 1992.

Transformations

Transformation may be by any known method for introducingpolynucleotides into a host cell, including packaging the polynucleotidein a virus and transducing a host cell with the virus, and by directuptake of the polynucleotide. The transformation procedures selecteddepends upon the host to be transformed. Bacterial transformation bydirect uptake generally employs treatment with calcium or rubidiumchloride. Cohen, Proc. Natl. Acad. Sci. USA 69:2110 (1972). Yeasttransformation by direct uptake may be conducted using the calciumphosphate precipitation method of Graham et al., Virology 52:526 (1978),or modification thereof.

Vector Construction

Vector construction employs methods known in the art. Generally,site-specific DNA cleavage is performed by treating with suitablerestriction enzymes under conditions which generally are specified bythe manufacturer of these commercially available enzymes. Usually, about1 microgram (μg) of plasmid or DNA sequence is cleaved by 1 unit ofenzyme in about 20 μl of buffer solution by incubation at 37° C. for 1to 2 hours. After incubation with the restriction enzyme, protein isremoved by phenol/chloroform extraction and the DNA recovered byprecipitation with ethanol. The cleaved fragments may be separated usingpolyacrylamide or agarose gel electrophoresis methods, according tomethods known by the routineer.

Sticky end cleavage fragments may be blunt ended using E. coli DNApolymerase 1 (Klenow) in the presence of the appropriate deoxynucleotidetriphosphates (dNTPs) present in the mixture. Treatment with S1 nucleasealso may be used, resulting in the hydrolysis of any single stranded DNAportions.

Ligations are performed using standard buffer and temperature conditionsusing T4 DNA ligase and ATP. Sticky end ligations require less ATP andless ligase than blunt end ligations. When vector fragments are used aspart of a ligation mixture, the vector fragment often is treated withbacterial alkaline phosphatase (BAP) or calf intestinal alkalinephosphatase to remove the 5'-phosphate and thus prevent religation ofthe vector. Or, restriction enzyme digestion of unwanted fragments canbe used to prevent ligation. Ligation mixtures are transformed intosuitable cloning hosts such as E. coli and successful transformantsselected by methods including antibiotic resistance, and then screenedfor the correct construction.

Construction of Desired DNA Sequences

Synthetic oligonucleotides may be prepared using an automatedoligonucleotide synthesizer such as that described by Warner, DNA 3:401(1984). If desired, the synthetic strands may be labelled with ³² P bytreatment with polynucleotide kinase in the presence of ³² P-ATP, usingstandard conditions for the reaction. DNA sequences including thoseisolated from genomic or cDNA libraries, may be modified by knownmethods which include site directed mutagenesis as described by Zoller,Nucleic Acids Res. 10:6487 (1982). Briefly, the DNA to be modified ispackaged into phage as a single stranded sequence, and converted to adouble stranded DNA with DNA polymerase using, as a primer, a syntheticoligonucleotide complementary to the portion of the DNA to be modified,and having the desired modification included in its own sequence.Culture of the transformed bacteria, which contain replications of eachstrand of the phage, are plated in agar to obtain plaques.Theoretically, 50% of the new plaques contain phage having the mutatedsequence, and the remaining 50% have the original sequence. Replicatesof the plaques are hybridized to labelled synthetic probe attemperatures and conditions suitable for hybridization with the correctstrand, but not with the unmodified sequence. The sequences which havebeen identified by hybridization are recovered and cloned.

Hybridization With Probe

HBV genomic or DNA libraries may be probed using the procedure describedby Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 73:3961 (1975).Briefly, the DNA to be probed is immobilized on nitrocellulose filters,denatured and prehybridized with a buffer which contains 0-50%formamide, 0.75 M NaCl, 75 mM Na citrate, 0.02% (w/v) each of bovineserum albumin (BSA), polyvinyl pyrollidone and Ficoll, 50 mM NaPhosphate (pH 6.5), 0.1% SDS and 100 μg/ml carrier denatured DNA. Thepercentage of formamide in the buffer, as well as the time andtemperature conditions of the prehybridization and subsequenthybridization steps depends on the stringency required. Oligomericprobes which require lower stringency conditions are generally used withlow percentages of formamide, lower temperatures, and longerhybridization times. Probes containing more than 30 or 40 nucleotidessuch as those derived from genomic sequences generally employ highertemperatures, for example, about 40 to 42° C., and a high percentage,for example, 50% formamide. Following prehybridization, a ³² P-labelledoligonucleotide probe is added to the buffer, and the filters areincubated in this mixture under hybridization conditions. After washing,the treated filters are subjected to autoradiography to show thelocation of the hybridized probe. DNA in corresponding locations on theoriginal agar plates is used as the source of the desired DNA.

Verification of Construction and Sequencing

For standard vector constructions, ligation mixtures are transformedinto E. coli strain HB101 or other suitable host, and successfultransformants selected by antibiotic resistance or other markers.Plasmids from the transformants then are prepared according to themethod of Clewell et al., Proc. Natl. Acad. Sci. USA 62:1159 (1969)usually following chloramphenicol amplification as reported by Clewellet al., J. Bacteriol. 110:667 (1972). The DNA is isolated and analyzedusually by restriction enzyme analysis and.or sequencing. Sequencing maybe by the well-known dideoxy method of Sanger et al., Proc. Natl. Acad.Sci. USA 74:5463 (1977) as further described by Messing et al., NucleicAcid Res. 9:309 (1981), or by the method reported by Maxam et al.,Methods in Enzymology 65:499 (1980). Problems with band compression,which are sometimes observed in GC rich regions, are overcome by use ofT-deazoguanosine according to the method reported by Barr et al.,Biotechniques 4:428 (1986).

Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay (ELISA) can be used to measure eitherantigen or antibody concentrations. This method depends upon conjugationof an enzyme label to either an antigen or antibody, and uses the boundenzyme activity (signal generated) as a quantitative label (measurablegenerated signal). Methods which utilize enzymes as labels are describedherein, as are examples of such enzyme labels.

Preparation of Mutant HBV Nucleic Acid Sequences

The source of the mutant HBV agent is an individual or pooled plasma,serum or liver homogenate from a human or chimpanzee infected with themutant HBV virus meeting the clinical and laboratory criteria describedherein. A chimpanzee alternatively can be experimentally infected withblood from another individual with mutant HBV hepatitis meeting thecriteria described hereinbelow. A pool can be made by combining manyindividual plasma, serum or liver homogenate samples containing highlevels of alanine transferase activity; this activity results fromhepatic injury due to mutant HBV infection.

For example, a nucleic acid library from plasma, serum or liverhomogenate, preferably but not necessarily high titer, is generated asfollows. First, viral particles are isolated from the plasma, serum orliver homogenate; then an aliquot is diluted in a buffered solution,such as one containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl.Debris is removed by centrifugation, for example, for 20 minutes at15,000×g at 20° C. Viral particles in the resulting supernatant then arepelleted by centrifugation under appropriate conditions which can bedetermined routinely by one skilled in the art. To release the viralgenome, the particles are disrupted by suspending the pellets in analiquot of an SDS suspension, for example, one containing 1% SDS, 120 mMEDTA, 10 mM Tris-HCl , pH 7.5, which also contains 2 mg/ml proteinase K,which is followed by incubation at appropriate conditions, for example,45° C. for 90 minutes. Nucleic acids are isolated by adding, forexample, 0.8 μg MS2 bacteriophage RNA as carrier, and extracting themixture four times with a 1:1 mixture of phenol:chloroform (phenolsaturated with 0.5M Tris-HCl, pH 7.5, 0.1% (v/v) beta-mercaptoethanol,0.1% (w/v) hydroxyquinolone, followed by extraction two times withchloroform. The aqueous phase is concentrated with, for example,1-butanol prior to precipitation with 2.5 volumes of absolute ethanolovernight at -20° C. Nucleic acids are recovered by centrifugation in,for example, a Beckman SW41 rotor at 40,000 rpm for 90 min at 4° C., anddissolved in water that is treated with 0.05% (v/v) diethylpyrocarbonateand autoclaved.

Nucleic acid obtained by the above procedure is denatured with, forexample, 17.5 mM CH₃ HgOH; cDNA then is synthesized using this denaturednucleic acid as template, and is cloned into the EcoRI site of phagelambda-gt11, for example, by using methods described by Huynh (1985)supra, except that random primers replace oligo(dT) 12-18 during thesynthesis of the first nucleic acid strand by reverse transcriptase (seeTaylor et al., 1976!).

The lambda-gt11 genomic library generated thusly is screened forepitopes that can bind specifically with serum, plasma or a liverhomogenate from an individual who had previously experienced hepatitisdue to mutant Hepatitis B Virus (one which meets the criteria as setforth hereinbelow). About 10⁴ -10⁷ phage are screened with sera, plasma,or liver homogenates using the methods of Huyng et al. (supra). Boundhuman antibody can be detected with sheep anti-human Ig antisera that isradio-labeled with ¹²⁵ I or other suitable reporter molecules includingHRPO, alkaline phosphatase and others. Positive phages are identifiedand purified. These phages then are tested for specificity of binding tosera from a pre-determined number of different humans previouslyinfected with the mutant HBV agent, using the same method. Ideally, thephage will encode a polypeptide that reacts with all or a majority ofthe sera, plasma or liver homogenates that are tested, and will notreact with sera, plasma or liver homogenates from individuals who aredetermined to be "negative" according to the criteria set forth hereinfor the mutant HBV agent as well as hepatitis A, non-mutant B, C, and E.By following these procedures, a clone that encodes a polypeptide whichis specifically recognized immunologically by sera, plasma or liverhomogenates from non-A, non-mutant B, non-C, non-E-identified patients.

The present invention will now be described by way of Examples.

EXAMPLES Example 1. Identification of Serum Samples

Initial Laboratory Determinations

Commercially available immunoassays (AUSZYME® and AUSRIA®II, AbbottLaboratories, Abbott Park, Ill.) were used for the determination ofantibody to HBsAg. Following manufacturer's directions, a patient testsample of serum was tested three times by the AUSZYME® assay forantibody to HBsAg. The AUSZYME® kit contains a solid phase (bead) uponwhich monoclonal antibodies to HBsAg have been coated. The patient testsample tested negative each of the three times for antibody to HBsAgusing this test kit. The patient test sample was retested using theAUSRIA®II test kit, which contains a polyclonal antibody to Hepatitis Bsurface antigen. The results of the AUSRIA®II test indicated that thepatient was positive for antibody to HBsAg.

Further Laboratory Testing

A serum test sample from the patient that had tested negative in theAUSZYME® monoclonal antibody test for antibody to HBsAg and positive inthe AUSRIA®II polyclonal antibody test for antibody to HBsAg was diluted1:20. Cross-exchange of AUSRIA®II reagents and AUSZYME® monoclonalreagents was performed (i.e., AUSRIA®II beads with AUSZYME® monoclonalantibody label, and vice versa). The preferred test conditions forevaluation were overnight incubation of the test sample at roomtemperature for the first incubation and four hours, room temperaturefor the second incubation. The test sample also was diluted serially,and dilutions were tested and compared with similar dilutions (5-fold)of the positive control. Data from these tests are presented below inTable 1.

                  TABLE 1    ______________________________________    AUSRIA ® II .sup.125 I* Antibody                       AUSZYME ® HRP*-Antibody                Auszyme               Auszyme    Ausria II Beads                Beads      Ausria II Beads                                      Beads    × cpm             S/C    × cpm                            S/C  × 492                                       S/C  × 492                                                  S/C    ______________________________________    NC   138     --     84    --   .008  --   .003  --    PC   13,782  47.5   12,548                              71.1 .522  9.0  2.410 45.5    Sam- 5,587   20.2   408   2.3  .087  1.5  .016  0.3    ple    ______________________________________     *.sup.125 I = radioactive I labelled antibody; HRP = Horseradish     peroxidase labeled antibody

The data previously indicated that the test sample was reactive withAUSRIA®II but not with AUSZYME® monoclonal. The data from Table 1 showthat when the cross-exchange of reagents were used for the test sampletesting, the test sample was weakly reactive with both combinations, butwas somewhat more reactive with AUSZYME® beads and ¹²⁵ I-antibody thanwith AUSRIA®II beads and AUSZYME®-HRP antibody. Further investigation ofthe differences between the antibodies from each test system indicatedthat the main reactant in the AUSZYME® is an anti-a antibody, as is theHRP-antibody. In AUSRIA®II, the bead is coated with a polyclonalantibody anti-HBs having a strong anti-a, but with anti-d and anti-ycapabilities. Further, the ¹²⁵ I-antibody in AUSRIA®II is a polyclonalwhich is predominantly anti-a, but both bead and probe antibodies areconsidered to be broad spectrum. The monoclonal antibody with itsfocused specificity on anti-a was not reactive with the test sample.Although the test sample reacted with the polyclonal antisera, it wasthought that the reactivity of the polyclonal antibody would be alteredsince it too predominantly detected anti-a. A dilution curve withAUSRIA®II antibody (not shown) revealed that it was in fact altered,although the broad spectrum of the polyclonal reagent was adequate todetect the anti-HBs present in the test sample.

Example 2. Determination of Sequence

Since it was believed that the sample was a form of a mutant HBV,sequencing was performed to determine if any variables were present inthe nucleotide and amino acid sequence of HBV surface antigen. TheSanger dideoxy sequencing reaction, as described in T. Maniatis et al.,Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y., Book 2: Chapter 13, 13.1-13.77 (1989)was performed. Results of sequence determinations showed a two aminoacid insertion in the amino acid sequence of the HBsAg at position 122.The nucleotide and amino acid sequence of the mutant HBV are presentedin SEQUENCE ID NO.1 and SEQUENCE ID NO.2. Repetition of the methodrevealed slight amino acid changes, and are presented as SEQUENCE IDNO.3 and SEQUENCE ID. NO.4. What each amino acid sequence has in commonis the two amino acid (N-T) insertion at position 122 of HBsAg. Thusthere is present in the sequence of HBsAg a modification in which thereis an insertion of two amino acids at position 122 (N-T), whichmodification corresponds to a six nucleotide insertion at position 366of the HBsAg genome.

The present invention thus provides reagents and methods for determiningthe presence of mutant HBsAg in a test sample. It is apparent thatassaying with a monoclonal antibody test which predominantly utilizeanti-a antibodies will not detect this particular mutant Further, it ispredictable based on the data presented that current vaccines will notbe protective against this mutant strain of HBV. Thus, reagents of thusmutant HBV are useful for detection of HBV in test samples, and also,for vaccine production. It is contemplated and within the scope of thepresent invention that a polynucleotide or polypeptide specific for themutant HBV described herein, or antibodies produced from thesepolypeptides and polynucleotides, can be combined with present assayreagents and incorporated into current assay procedures for thedetection of antibody to HBsAg. Alternatively, these polynucleotide orpolypeptide specific for the mutant HBV described herein, or antibodiesproduced from these polypeptides and polynucleotides, can be usedseparately for detection of the mutant strain of HBV in which the "a"determinant has a two amino acid insertion at position 122 of the HBsAgsequence.

Other uses or variations of the present invention will be apparent tothose of ordinary skill of the art when considering this disclosure.Therefore, the present invention is intended to be limited only by theappended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 4    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 684 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 1..684    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - ATG GAG AAC ACC ACA TCA GGA CTC CTA GGA CC - #C CTG CTC GTG TTA CAG      48    Met Glu Asn Thr Thr Ser Gly Leu Leu Gly Pr - #o Leu Leu Val Leu Gln    #                15    - GCG GGG TTT TTC TTG TTG ACA AAA ATC CTC AC - #A ATA CCA CAG AGT CTA      96    Ala Gly Phe Phe Leu Leu Thr Lys Ile Leu Th - #r Ile Pro Gln Ser Leu    #            30    - GAC TCG TGG TGG ACT TCT CTC AGT TTT CTA GG - #G GGA ACA CCC GTG TGT     144    Asp Ser Trp Trp Thr Ser Leu Ser Phe Leu Gl - #y Gly Thr Pro Val Cys    #        45    - TCT GGC CAA AAT TCG CAG TCC CAA ATC TCC AG - #T CAC TCA CCA AAC TGC     192    Ser Gly Gln Asn Ser Gln Ser Gln Ile Ser Se - #r His Ser Pro Thr Cys    #    60    - TGT CCT CCA ATT TGT CCT GGT TAT CGC TGG AT - #G TGT CTG CGG CGT TTT     240    Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Me - #t Cys Leu Arg Arg Phe    #80    - ATC ATC TTC CTC TGC ATC CTG CTG CTA TGC CT - #C ATC TTC TTG TTG GTT     288    Ile Ile Phe Leu Cys Ile Leu Leu Leu Cys Le - #u Ile Phe Leu Leu Val    #                95    - CCT CTG GAC TAC CAA GGT ATG TTG CCC GTT TG - #T CCT CTA ATT CCA GGA     336    Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cy - #s Pro Leu Ile Pro Gly    #           110    - TCA TCA ACA ACC AGC ACC GGA CCA TGC AGG AA - #C ACA ACC TGC ACG ACT     384    Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg As - #n Thr Thr Cys Thr Thr    #       125    - CCT GCT CAA GGA ACC TCT ATG TTT CCC TCA TG - #T TGC TGT ACA AAA CCT     432    Pro Ala Gln Gly Thr Ser Met Phe Pro Ser Cy - #s Cys Cys Thr Lys Pro    #   140    - ACC GAC AGA AAC TGC ACC TGT ATT CCC ATC CC - #A TCA TCT TGG GCT TTC     480    Thr Asp Arg Asn Cys Thr Cys Ile Pro Ile Pr - #o Ser Ser Trp Ala Phe    145                 1 - #50                 1 - #55                 1 -    #60    - GCA AAA TTC CTA TGG GAG TGG GCC TCA GTC CG - #T TTC TCT TGG CTC AGT     528    Ala Lys Phe Leu Trp Glu Trp Ala Ser Val Ar - #g Phe Ser Trp Leu Ser    #               175    - TTA CTA GTG CCA TTT GTT CAG TGG TTC GTA GG - #G CTT TCC CCC ACT GTC     576    Leu Leu Val Pro Phe Val Gln Trp Phe Val Gl - #y Leu Ser Pro Thr Val    #           190    - TGG CTT TCA GTT ATA TGG ATG ATG TGG TAT TG - #G GGG CCA AGT CTG TAC     624    Trp Leu Ser Val Ile Trp Met Met Trp Tyr Tr - #p Gly Pro Ser Leu Tyr    #       205    - AAC ATC TTG AGT CCC TTT ATG CCG CTG TTA CC - #A ATT TTC TAT TGT CTT     672    Asn Ile Leu Ser Pro Phe Met Pro Leu Leu Pr - #o Ile Phe Tyr Cys Leu    #   220    #      684    Trp Val Tyr Ile    225    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 228 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Met Glu Asn Thr Thr Ser Gly Leu Leu Gly Pr - #o Leu Leu Val Leu Gln    #                15    - Ala Gly Phe Phe Leu Leu Thr Lys Ile Leu Th - #r Ile Pro Gln Ser Leu    #            30    - Asp Ser Trp Trp Thr Ser Leu Ser Phe Leu Gl - #y Gly Thr Pro Val Cys    #        45    - Ser Gly Gln Asn Ser Gln Ser Gln Ile Ser Se - #r His Ser Pro Thr Cys    #    60    - Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Me - #t Cys Leu Arg Arg Phe    #80    - Ile Ile Phe Leu Cys Ile Leu Leu Leu Cys Le - #u Ile Phe Leu Leu Val    #                95    - Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cy - #s Pro Leu Ile Pro Gly    #           110    - Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg As - #n Thr Thr Cys Thr Thr    #       125    - Pro Ala Gln Gly Thr Ser Met Phe Pro Ser Cy - #s Cys Cys Thr Lys Pro    #   140    - Thr Asp Arg Asn Cys Thr Cys Ile Pro Ile Pr - #o Ser Ser Trp Ala Phe    145                 1 - #50                 1 - #55                 1 -    #60    - Ala Lys Phe Leu Trp Glu Trp Ala Ser Val Ar - #g Phe Ser Trp Leu Ser    #               175    - Leu Leu Val Pro Phe Val Gln Trp Phe Val Gl - #y Leu Ser Pro Thr Val    #           190    - Trp Leu Ser Val Ile Trp Met Met Trp Tyr Tr - #p Gly Pro Ser Leu Tyr    #       205    - Asn Ile Leu Ser Pro Phe Met Pro Leu Leu Pr - #o Ile Phe Tyr Cys Leu    #   220    - Trp Val Tyr Ile    225    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 228 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Met Glu Asn Thr Thr Ser Gly Leu Leu Gly Pr - #o Leu Leu Val Leu Gln    #                15    - Ala Gly Phe Phe Leu Leu Thr Lys Ile Leu Th - #r Ile Pro Gln Ser Leu    #            30    - Asp Ser Trp Trp Thr Ser Leu Ser Phe Leu Gl - #y Gly Thr Pro Val Cys    #        45    - Phe Gly Gln Asn Ser Gln Thr Gln Ile Ser Se - #r His Ser Pro Thr Cys    #    60    - Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Me - #t Cys Leu Arg Arg Phe    #80    - Ile Ile Phe Leu Cys Ile Leu Leu Leu Cys Le - #u Ile Phe Leu Leu Val    #                95    - Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cy - #s Pro Leu Ile Pro Gly    #           110    - Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg As - #n Thr Thr Cys Thr Thr    #       125    - Pro Ala Gln Gly Thr Ser Met Phe Pro Ser Cy - #s Cys Cys Thr Lys Pro    #   140    - Thr Asp Arg Asn Cys Thr Cys Ile Pro Ile Pr - #o Ser Ser Trp Ala Phe    145                 1 - #50                 1 - #55                 1 -    #60    - Val Lys Phe Leu Trp Glu Trp Ala Ser Val Ar - #g Phe Ser Trp Leu Ser    #               175    - Phe Leu Val Pro Ile Val Gln Trp Phe Ala Gl - #y Leu Ser Pro Thr Val    #           190    - Trp Leu Ser Val Ile Trp Met Met Trp Tyr Tr - #p Gly Pro Ser Leu Tyr    #       205    - Asn Ile Leu Ser Pro Phe Met Pro Leu Leu Pr - #o Ile Phe Tyr Cys Leu    #   220    - Trp Val Tyr Ile    225    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 228 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Met Glu Asn Thr Thr Ser Gly His Leu Thr Hi - #s Leu Leu Val Leu Gln    #                15    - Ala Gly Phe Phe Leu Leu Thr Lys Ile Leu Th - #r Ile Pro Gln Ser Leu    #            30    - Asp Ser Trp Trp Thr Ser Leu Ser Phe Leu Gl - #y Gly Thr Pro Val Cys    #        45    - Ser Gly Gln Asn Ser Gln Ser Gln Ile Ser Se - #r His Ser Pro Thr Cys    #    60    - Ser Pro Pro Ile Cys Pro Gly Tyr Arg Trp Me - #t Cys Leu Arg Arg Phe    #80    - Ile Ile Phe Leu Cys Ile Leu Leu Leu Cys Le - #u Ile Phe Leu Leu Val    #                95    - Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cy - #s Pro Leu Ile Pro Gly    #           110    - Ser Ser Thr Thr Ser Thr Gly Pro Cys Arg As - #n Thr Thr Cys Thr Thr    #       125    - Pro Ala Gln Gly Thr Ser Met Phe Pro Ser Cy - #s Cys Cys Thr Lys Pro    #   140    - Thr Asp Arg Asn Cys Thr Cys Leu Pro Ile Pr - #o Ser Ser Trp Ala Phe    145                 1 - #50                 1 - #55                 1 -    #60    - Ala Lys Phe Leu Trp Glu Trp Ala Ser Val Gl - #y Phe Ser Trp Leu Ser    #               175    - Leu Leu Val Pro Phe Val Gln Trp Phe Val Gl - #y Phe Pro Pro Thr Val    #           190    - Trp Leu Ser Val Ile Trp Met Met Trp Tyr Tr - #p Gly Pro Ser Leu Tyr    #       205    - Asn Ile Leu Ser Pro Phe Met Pro Leu Leu Pr - #o Ile Phe Tyr Cys Leu    #   220    - Trp Val Tyr Ile    225    __________________________________________________________________________

What is claimed is:
 1. A monoclonal antibody which specifically binds to mutant hepatitis B virus surface antigen (HBsAg) "a" determinant and does not cross react with native HBsAg "a" determinant, wherein the mutation of said mutant "a" determinant is insertion of the amino acid sequence Asn-Thr between positions 122 and 123 of the HBsAg sequence.
 2. A kit for determining the presence of mutant hepatitis B surface antigen or antibody, comprising a container containing said antibody of claim
 1. 3. The kit of claim 2 wherein said antibody is attached to a solid phase.
 4. A method for detecting mutant hepatitis B surface antigen (HBsAg) "a" determinant in a test sample, comprising:a. reacting a test sample suspected of containing mutant HBsAg with the antibody of claim 1, for a time and under conditions sufficient to allow the formation of antibody/antigen complexes; and b. detecting said complexes containing the antibody.
 5. The method of claim 4 wherein said HBsAg encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
 6. The method of claim 4, wherein said antibody/antigen complexes are contacted with an indicator reagent for a time and under conditions sufficient to allow the formation of antibody/antigen/indicator reagent complexes to form before. 